Made available in DSpace on 2014-08-20T14:37:54Z (GMT). No. of bitstreams: 1 dissertacao_caroline_bohnen_de_matos.pdf: 562109 bytes, checksum: 6339bc1f54f1654dae0b8adf8c276724 (MD5) Previous issue date: 2014-02-12 / Sporotrichosis is the main domestic feline ringworm, highly relevant because it is a zoonosis involving six fungal species belonging to the Sporothrix complex. The fungal isolation of home and veterinary environments has been proven, proving to be an important source of infection for animals and humans. The search for alternative to disinfectants commonly used products is increasing, mainly by continuing reports of resistance by microorganisms. The Origanum vulgare and Rosmarinus officinalis stand out because they have proven antimicrobial activity. Given the above, the objective of the study was to evaluate the efficacy of disinfectants and plant extracts in eliminating the fungus Sporothrix complex, and report a case of multifactorial dermatitis in a dog. For this, in vitro tests were performed using the technique of direct exposure to evaluate the action of the tincture, aqueous extract and alcoholic extract of O. vulgare and R. officinalis against clinical isolates of dogs and cats with sporotrichosis. Subsequently, the plant extracts that showed better results in the direct exposure test were evaluated for the elimination of Sporothrix spp different contaminated surfaces, also using different disinfectants. As an experimental model surfaces of stainless steel, formica and ceramic tile floors, tested in quadruplicate using the technique of spray-wipe-spray were used. According to the study, the hydroalcoholic extract and tincture of O. vulgare and R. officinalis were effective in eliminating the fungus Sporothrix complex, with no difference from the area used, including showing better results when compared to commercial products used. / A esporotricose é a principal micose dos felinos domésticos, apresentando grande importância por tratar-se de uma zoonose envolvendo seis espécies fúngicas pertencem ao complexo Sporothrix. O isolamento fúngico de ambientes domiciliares e veterinários já foi comprovado, revelando-se uma importante fonte de infecção para animais e humanos. A busca de produtos alternativos aos desinfetantes utilizados normalmente é crescente, principalmente pelos constantes relatos de resistência por parte dos microrganismos. O Origanum vulgare e o Rosmarinus officinalis destacam-se por apresentarem atividade antimicrobiana comprovada. Diante do exposto, o objetivo do estudo foi avaliar a eficácia de desinfetantes e de extratos vegetais na eliminação de fungos do complexo Sporothrix, e relatar um caso clínico de dermatite multifatorial em um canino. Para isso, testes in vitro foram realizados através da técnica de exposição direta para avaliar a ação da tintura, do extrato aquoso e do extrato hidroalcoólico de O. vulgare e R. officinalis frente a isolados clínicos de cães e gatos com esporotricose. Posteriormente, os extratos vegetais que demonstraram melhores resultados no teste de exposição direta foram avaliados em relação a eliminação do Sporothrix spp de diferentes superfícies contaminadas, utilizando também diferentes desinfetantes. Como modelo experimental foram utilizadas superfícies de aço inoxidável, fórmica e piso cerâmico, testadas em quadruplicata através da técnica de spray-wipe-spray. De acordo com o estudo, o extrato hidroalcoolico e a tintura de O. vulgare e R. officinalis foram eficazes na eliminação de fungos do complexo Sporothrix, não havendo diferença em relação a superfície utilizada, inclusive apresentando melhores resultados quando comparado aos produtos comerciais utilizados.

L'awara (Astrocaryum vulgare M.) est le fruit d'un palmier utilisé principalement au niveau alimentaire pour sa pulpe riche en huile. L'objectif de ce travail a été de caractériser chimiquement l'huile de pulpe d'awara et d'évaluer ses propriétés anti‑inflammatoires dans différents modèles expérimentaux. L'huile de pulpe, dont les acides gras principaux sont l'acide oléique et palmitique, a montré une forte teneur en caroténoïdes. Les microconstituants identifés sont principalement représentés par le β‑carotène (caroténoïdes), le β‑sitostérol, l'arundoin (phytostérols) et l'α‑tocopérol. L'huile de pulpe a montré des effets anti‑inflammatoires dans un modèle in vivo de choc endotoxique par inhibition de la production de cytokines pro‑inflammatoires (tumor necrosis factor α (TNFα), interleukin (IL)‑6) et augmentation de la production d'une cytokine anti‑inflammatoire (IL‑10). Ces effets anti‑inflammatoires ont été confirmés dans un modèle in vivo d'inflammation bronchique. L'huile de pulpe a diminué l'afflux de cellules inflammatoires au niveau pulmonaire, principalement d'éosinophiles et de lymphocytes. La fraction insaponifiable a, par la suite, montré des effets inhibiteurs sur la production de différents médiateurs inflammatoires (oxyde nitrique, prostaglandine E2, TNFα, IL‑6, IL‑10) ainsi que sur l'expression d'enzymes induites lors de l'inflammation (iNOS et COX‑2) dans un modèle in vitro de macrophages activés. La fraction insaponifiable a également montré un effet inhibiteur sur la production de cytokines (TNFα, d'IL‑6 et d'IL‑10) dans le modèle in vivo de choc endotoxique. Ces résultats confirment l'implication des microconstituants de l'huile de pulpe d'awara dans ses effets antioxydants et anti‑inflammatoires et suggèrent un rôle préventif et/ou thérapeutique de l'huile de pulpe d'awara et de sa fraction insaponifiable dans les pathologies associées à l'inflammation. / Awara (Astrocaryum vulgare M.) is a palm fruit mainly used for nutritional purpose because of its oily pulp. The aim of this study was to characterize awara pulp oil and evaluate its anti‑inflammatory properties in different experimental models. The pulp oil, whose major fatty acids are oleic and palmitic acid, showed a high content of carotenoids. The minor compounds identified are mainly represented by β‑carotene (carotenoids), β‑sitosterol, arundoin (phytosterols) and α‑tocopérol. The pulp oil showed anti‑inflammatory effects in an in vivo model of endotoxic shock by inhibiting the production of proinflammatory cytokines (tumor necrosis factor α (TNFα), interleukin (IL)‑6) and increasing the production of an anti‑inflammatory cytokine (IL‑10). These anti‑inflammatory effects have been confirmed in an in vivo model of airway inflammation. The pulp oil has reduced the influx of inflammatory cells in the lungs, mainly of eosinophils and lymphocytes. The unsaponifiable fraction has subsequently shown inhibitory effects on the production of various inflammatory mediators (nitric oxide, prostaglandin E2, TNFα, IL‑6, IL‑10) and on the expression of enzymes induced during inflammation (iNOS and COX‑2) in an in vitro model of activated macrophages. The unsaponifiable fraction also showed an inhibitory effect on cytokine production (TNFα, IL‑6 and IL‑10) in endotoxic shock model. These results confirm the role of micronutrients of awara pulp oil in its anti‑inflammatory properties and suggest a preventive and/or therapeutic role of awara pulp oil and its unsaponifiable fraction in inflammation‑related diseases.

Die Verfügbarkeit des Makroelementes Phosphor ist für Lebewesen eingeschränkt. Besonders bei der Pflanzenproduktion und der Tierernährung spielt die Phosphorverfügbarkeit eine wichtige Rolle bei der ökonomischen Nutzung der Ressourcen. In den Fokus der Wissenschaft zur Lösung des Phosphorproblems gerieten die Phytasen, da monogastrische Tiere nicht in der Lage sind das in den Pflanzensamen gespeicherte Phytat zu nutzen. Die gentechnische Veränderung von Pflanzen stellt eine effiziente Möglichkeit zur Produktion von phosphatfreisetzenden Enzymen, zur Erhöhung der Biomasseproduktion und zur Veränderung der Inhaltstoffe dar. In dieser Arbeit wurden erfolgreich transgene Pflanzen der Arten Nicotiana tabacum L. cv. Samsun und Hordeum vulgare L. cv. Golden Promise erzeugt, die in der Lage waren die Phytase aus Klebsiella sp. ASR1 bzw. aus Bacillus amyloliquefaciens FZB45 zu produzieren. Es wurde für jedes Protein eine Strategie zur Reinigung des aktiven Enzyms aus den verschiedenen Wirtsorganismen entwickelt und seine biochemischen Eigenschaften charakterisiert. Es wurde gezeigt, dass die β-Propeller-Phytase aus Bacillus im Gegensatz zur sauren Phytase aus Klebsiella durch die posttranslationale Modifikation teilweise ihre Eigenschaften ändert. Die Auswirkungen der heterologen Expression der Phytasen auf die Veränderung der Anteile von Phytinsäure und anorganischem Phosphor, in Relation zum gesamten Phosphor, in den Gerstensamen wurden untersucht. Es wurde eine Reduktion des Phytinsäuregehaltes um 19 % und eine Erhöhung des Gehalts anorganischem Phosphor zwischen 27 % und 78 nachgewiesen. Mit Hilfe von spezifischen Signalsequenzen gelang es die Phytaseproteine aus der Wurzel in das umgebende Medium zu sekretieren. Die Sekretion der Bacillus Phytase führte zu einer Steigerung der Biomasseproduktion von Nicotiana tabacum L. unter unsterilen Wachstumsbedingungen mit Phytat als einziger P-Quelle um 34 %. / Due to the bad availability of phosphorus in natural habitats the improvement of phosphorus accessibility to organisms became an important topic of research, particularly for agriculture and animal nutrition. In plant seeds phosphorus is bound to D-myo-inositol to form phytic acid that is indigestible for mono gastric animals. Therefore the use of phytases to hydrolyze the phytic acid and to mobilize the anorganic phosphorus came in focus to science. Genetic engineering gave the opportunity to improve the phosphorus availability. Genetic manipulation of plants is a suitable tool to produce phosphorus releasing enzymes, thereby increasing the biomass and decreasing the content of phytic acid in plants. In this work I generated transgenic plants of Nicotiana tabacum and Hordeum vulgare vulgare which successfully express the phytase gene phyK from Klebsiella pneumonia ASR1 and phyC from Bacillus amyloliquefaciens FZB42. Chromatographically purification strategies were developed and biochemical properties were characterized for all phytase proteins. All enzymes were active and PhyC was posttranslational modified. The effect of the recombinant phytase activity to total phosphorus, phytic acid and anorganic phosphorus content of barley seeds were elucidated. The phyK-expression in barley seeds yield a reduction of phytic acid content of about 19 % and an increase of anorganic phosphorus of about 78 %. Transgenic barley seeds with phyC gene expression show an increase of anorganic phosphorus content from 27 % to 48 % but no decrease in phytic acid content. In growth experiments no special phenotypes of plant containing the recombinant protein were visible. The insertion of apoplastic signal sequences in front of the phytase genes resulted in secretion of the proteins into the rhizosphere. The secretion of PhyC led to an improved growth of Nicotiana tabacum under unsterile conditions with sodium phytate as single phosphorus source increasing the biomass up to 34 %.

Made available in DSpace on 2017-07-25T19:30:53Z (GMT). No. of bitstreams: 1 Camila Ferreiratese.pdf: 1360081 bytes, checksum: 4c38254bbea500111258b4b71abc497f (MD5) Previous issue date: 2015-11-26 / Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná / The arrangement of plants in paired spacing has been used in some cultivars of barley with good yield results. However, this still presents divergent results spacing between the work already carried out. In addition, it is necessary to adjust the rate of fertilizer and plant population to be used in paired spacing, as compared to simple spacing. The objective of this work was to evaluate rates of fertilizer at seeding and populations of plants in paired spacing in comparison with the simple in crop of barley. In this sense, two experiments were installed, differing them by cultivating, in farm school of the Universidade Estadual de Ponta Grossa, in the municipality of Ponta Grossa, PR, in the year 2013, with repetition in the year 2014. The experimental design adopted in two years was randomized in main plots plots scheme, with four replicates. The treatments consisted of plots with two spatial arrangements of plants (simple and paired spacing), subplots with two doses of fertilizer applied at seeding (100 and 150 kg ha-1) and subsubparcelas with four plant populations (270, 360, 540 and 450 seeds m-2). The treatments were applied in two barley cultivars, BRS-Cauê and BRS-Elis. With the results obtained shows that the paired spacing does not present positive results consolidated in crop of barley, once the simple spacing produced more than the paired. The largest rate of fertilizer applied at seeding should be adopted regardless of the spatial arrangement is used, because it resulted in higher yield in one of the cultivars in the two years of research. The recommended seeding density for the crop of barley (250 to 300 plants m-2) should be kept regardless of the spatial arrangement adopted, since the elevation of density not provided positive results that justify their adoption. / O arranjo de plantas em espaçamento pareado vem sendo utilizado em algumas cultivares de cevada com bons resultados de produtividade. No entanto, esse espaçamento ainda apresenta resultados divergentes entre os trabalhos já realizados. Além disso, é necessária a readequação da dose de fertilizante e da população de planta a serem utilizadas no espaçamento pareado, em comparação ao espaçamento simples. O objetivo do trabalho foi avaliar doses de fertilizante na semeadura e populações de plantas no espaçamento pareado em comparação com o simples na cultura da cevada. Nesse sentido, foram instalados dois experimentos, diferindo-os pelo cultivar, na Fazenda Escola da Universidade Estadual de Ponta Grossa, no município de Ponta Grossa, PR, no ano de 2013, com repetição no ano de 2014. O delineamento experimental adotado nos dois anos foi de blocos ao acaso, em esquema de parcelas subsubdivididas, com quatro repetições. Os tratamentos constaram de parcelas com dois arranjos espaciais de plantas (espaçamento simples e pareado), de subparcelas com duas doses de fertilizante aplicado na semeadura (100 e 150 kg ha-1) e de subsubparcelas com quatro populações de plantas (270, 360, 450 e 540 sementes m-2). Os tratamentos foram aplicados em dois cultivares de cevada, BRS-Cauê e BRS-Elis. Com os resultados obtidos evidencia-se que o espaçamento pareado não apresenta resultados positivos consolidados na cultura da cevada, uma vez que o espaçamento simples produziu mais que o pareado. A maior dose de fertilizante aplicada na semeadura deve ser adotada independentemente do arranjo espacial utilizado, pois a mesma resultou em maior produtividade em um dos cultivares nos dois anos de pesquisa. A densidade de semeadura recomendada para a cultura da cevada (250 a 300 plantas m-2) deve ser mantida independentemente do arranjo espacial adotado, uma vez que a elevação da densidade não proporcionou resultados positivos que justificasse sua adoção.

Résumé : Chez lorge, la dégradation des polysaccarides des parois cellulaires et de lamidon est de première importance dans le contexte de sa valorisation brassicole. La présente recherche doctorale a été entreprise dans le but didentifier des marqueurs de sélection utilisables lors de lamélioration génétique de lorge de printemps pour ses qualités brassicoles. Des gènes candidats ont dabord été choisis sur base de leur expression dans les tissus de la graine en germination. Leur position cartographique a été déterminée et située par rapport à des marqueurs QTL connus de la qualité brassicole. Exploitant des variétés cultivées de qualités contrastées, une recherche de polymorphismes nucléotidiques au niveau de ces gènes a été réalisée. Tous les gènes savèrent polymorphes, certaines des variations de séquence conduisant à des modifications de la structure primaire de la protéine encodée. Plusieurs caractères technologiques ont été retenus et définis chez les variétés étudiées, en exploitant les données de la littérature : la teneur en extrait, la viscosité, la teneur en β-glucanes du mout, latténuation finale apparente et le pouvoir diastasique. Plusieurs caractères biochimiques ont été étudiés chez ces mêmes variétés et ont ciblé quatre activités enzymatiques impliquées dans la dégradation des polysaccharides pariétaux et de réserve. Le pouvoir discriminant et la simplicité du test dactivité amylase, ainsi que sa relation avec des critères technologiques importants, en font un test prometteur en vue de faciliter la sélection variétale. La relation entre les sites polymorphes de lADN et les caractères technologiques et biochimiques identifient une soixantaine de sites dont les polymorphismes semblent associés à au moins lun des caractères. Sur tous les sites polymorphes, treize sites sont choisis pour leur potentiel en tant que marqueurs de sélection. Ils distinguent en effet un allèle favorable et un allèle défavorable pour plus dun critère brassicole. Ces treize sites sont localisés sur des gènes de (1-3,1-4)-β-glucanase, un gène de (1-3)-β-glucanase, un gène de (1-4)-β-xylan endohydrolase et un gène dα-amylase. La mise en évidence de ces allèles peut être réalisée par des tests PCR simples (« allèles spécifiques ») et relativement peu coûteux, dont six ont été mis au point au terme de la recherche./ Summary: In barley, the degradation of cell wall polysaccharides and starch is of utmost importance for its malting quality. The aim of this thesis is the identification of selection markers useful in the breeding of spring Barley cultivars for improved malting quality. Candidate genes were first chosen based on their expression profile in tissues of germinating seeds. Their mapping positions were determined and compared with known QTLs for malting quality. Varieties with contrasted malting qualities were searched for DNA polymorphisms for each of these genes. All genes proved to be polymorphic, some of the sequence variations leading to changes in the primary structure of the encoded protein. Technological traits were chosen and used to characterize the varieties based on literature data: extract yield, viscosity, β-glucan content of the wort, final apparent attenuation and diastatic power. Biochemical traits were also investigated on the same varieties and focused on four enzyme activities implicated in cell wall polysaccharides and starch degradation. The Discriminant power and ease of the amylase test, as well as its relation with technological traits, make it a promising selection test in breeding programs. The relationship between the DNA polymorphisms and biochemical and technological traits reveals around sixty polymorphisms displaying apparent relationships with at least one trait. Thirteen out of them were chosen for their potential as selection markers. They are located on (1-3,1-4)-β-glucanase genes, on one (1-3)-β-glucanase gene, on one (1-4)-β-xylan endohydrolase gene and on one α-amylase gene. Detection of these alleles can be achieved by simple and inexpensive PCR tests ( allele specific), and six assay protocols have been set up at the completion of this research.

Made available in DSpace on 2015-05-14T12:59:48Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 2450774 bytes, checksum: 5a8c6f7ae617f05957b253f97b3104cd (MD5) Previous issue date: 2012-02-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cryptococcus neoformans is yeast capable of causing infection in humans called cryptococcosis that primarily affects immunocompromised patients. The infection usually affects the central nervous system causing meningoencephalitis with a severe and fatal evolution, accompanying or not, lung injury, fungemia and secondary outbreaks on the skin, bones and kidneys. The drugs of choice for treatment are usually amphotericin B associated with 5-flucytosine, however, increasing resistance to antifungal agents available and toxicity caused by them lead the search for new antifungals, becoming natural products as focus of studies in the research of substances with antifungal properties. Thus, the study aimed to investigate the antifungal activity of essential oils of Coriandrum sativum L. (coriander) and Foeniculum vulgare Mill. (fennel) in vitro against 14 strains of Cryptococcus neoformans. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were determinated by the microdilution technique, and the effects of essential oils on the morphology and time-kill curve of C. neoformans were evaluated. Was found for the essential oil of C. sativum MIC= 64 μg/mL and MFC= 64 μg/mL, and for the essential oil of F. vulgare, MIC= 256 μg/mL and MFC= 512 μg/mL. There were no changes in the morphology of LM 310 and ICB 59 strains of the yeast when subjected to contact with essential oils. Has been observed a decrease in count of viable forms with increasing concentrations of essential oils. In the time-kill curve of C. neoformans, compared to the essential oil of C. sativum, was observed fungicidal effect (decrease ≥ 3 log10UFC/mL from the initial inoculation), at 24 hours, at MICx4 for the strain LM 310, and in MICx2 and MICx4, for the strain ICB 59. The essential oil of F. vulgare, for strain LM 310, began fungicidal activity at the time of 4 hours in MICx2 and MICx4, the MIC was fungicidal at 8 hours. The fungicidal activity for the strain ICB 59 began in 4 hours for all concentrations of essential oil (MIC, MICx2 and MICx4). The essential oils of C. sativum and F. vulgare has antifungal activity against strains of C. neoformans, therefore represent a possibility to become new products with antifungal potential in the treatment of cryptococcosis. However, there is need for further studies to search deeper aspects of the essential oils tested, so they can be used in therapy. / Cryptococcus neoformans é uma levedura capaz de provocar no homem uma infecção denominada criptococose que acomete principalmente pacientes imunocomprometidos. A infecção geralmente atinge o sistema nervoso central provocando meningoencefalite, de evolução grave e fatal, acompanhada ou não, de lesão pulmonar, fungemia e focos secundários na pele, ossos e rins. Os fármacos de escolha para seu tratamento são geralmente anfotericina B associada a 5-fluocitosina. Entretanto, o aumento da resistência aos antifúngicos disponíveis e a toxicidade provocada por eles levam a busca de novos antifúngicos, fazendo com que os produtos naturais sejam foco de pesquisas na busca de substâncias com propriedades antifúngicas. Desta forma, o estudo teve como objetivo investigar a atividade antifúngica dos óleos essenciais de Coriandrum sativum L. (coentro) e Foeniculum vulgare Mill. (funcho) in vitro sobre 14 cepas de Cryptococcus neoformans. Foram determinadas a Concentração Inibitória Mínima (CIM) e Concentração Fungicida Mínima (CFM), pela técnica da microdiluição, e avaliados os efeitos dos óleos essenciais sobre a micromorfologia e a curva de morte fúngica de C. neoformans. Foi encontrado para o óleo essencial de C. sativum CIM= 64 μg/mL e CFM= 64 μg/mL, e para o óleo essencial de F. vulgare, CIM= 256 μg/mL e CFM= 512 μg/mL. Não foram observadas alterações na micromorfologia das cepas ICB 59 e LM 310 da levedura quando submetidas ao contato com os óleos essenciais. Foi constatada, porém uma diminuição na contagem de formas viáveis com aumento das concentrações dos óleos essenciais. Na curva de morte microbiana causada pelo óleo essencial de C. sativum sobre a cepa de C. neoformans LM 310, observou-se um efeito fungicida (redução ≥3 log10UFC/mL a partir do inóculo inicial), no tempo de 24 horas, na CIMx4; e nas CIMx2 e CIMx4, para a cepa ICB 59. O óleo essencial de F. vulgare, para a cepa LM 310, iniciou sua atividade fungicida no tempo de 4 horas nas CIMx2 e CIMx4, a CIM foi fungicida a partir de 8 horas. A atividade fungicida para a cepa ICB 59 iniciou-se em 4 horas para todas as concentrações do óleo essencial (CIM, CIMx2 e CIMx4). Os óleos essenciais de C. sativum e F. vulgare apresentaram atividade antifúngica contra cepas de C. neoformans, portanto, representam uma possibilidade de tornarem-se novos produtos com potencial efeito antifúngico no tratamento da criptococose. Há, entretanto, necessidade de estudos posteriores que pesquisem aspectos mais aprofundados dos óleos essenciais testados, para que possam ser utilizados na terapêutica.

As propriedades terapêuticas das plantas medicinais são cada vez mais estudadas, principalmente devido aos crescentes casos de resistência antimicrobiana, como observado em cepas do Complexo Sporothrix. As plantas da família Lamiaceae são conhecidas por suas propriedades antifúngicas, entretanto, são escassos seus estudos contra agentes causadores da esporotricose. Devido ao potencial promissor dessas plantas, objetivou-se (1) avaliar a atividade anti-Sporothrix spp. in vitro de Origanum vulgare L. (orégano), Origanum majorana L. (manjerona) e Rosmarinus officinalis L. (alecrim) nas formas de óleos essenciais, extratos aquosos de infusão e decocção e extratos hidroalcoólicos contra isolados clínicos de Sporothrix spp. obtidos de casos clínicos de esporotricose humana, canina e felina; (2) avaliar os principais constituintes químicos presentes nos óleos essenciais das plantas; e (3) avaliar a atividade citotóxica in vitro. Extratos aquosos e hidroalcoólicos foram preparados a partir de partes aéreas das plantas. Em óleos essenciais, produtos comerciais e produtos extraídos de partes aéreas por hidrodestilação em Clevenger foram testados. Ambos óleos foram analisados quimicamente por cromatografia gasosa. Testes in vitro foram realizados pela técnica de Microdiluição em Caldo contra isolados clínicos de Sporothrix spp. oriundos de humanos, caninos e felinos, bem como ambiental nas fases leveduriforme (CLSIM27A3) e filamentosa (CLSI-M38A2), sendo testados entre 72 a 0.07 mg/mL. Os efeitos citotóxicos foram avaliados através do ensaio MTT em células VERO (78 a 5000 μg/mL). Na fase leveduriforme, os óleos essenciais extraído e comercial de orégano apresentaram atividade anti-Sporothrix spp. nas concentrações inibitórias mínimas (CIM) e concentrações fungicidas mínimas (CFM) de 36 a ≤2.25 mg/mL e de alecrim entre 72 a≤2.25 mg/mL, não havendo diferença estatística entre os tipos de óleos. Por sua vez, óleo comercial de manjerona apresentou CIM/CFM de 18 a ≤2.25 mg/mL. Na fase micelial, 100% dos isolados felinos e caninos foram sensíveis aos óleos essenciais de orégano (0.14 a ≤0.07 mg/mL), alecrim (18 a ≤0,07 mg/mL) e manjerona (4.5 a ≤0.07 mg/mL). Extratos aquosos e hidroalcoólicos de orégano apresentaram atividade inibitória e fungicida, respectivamente, em 100% e entre 90% a 35% dos isolados animais (40 a ≤0.07 mg/mL). Nas mesmas concentrações, o extrato hidroalcoólico de manjerona inibiu de 90% a 100% dos isolados. Entretanto, os demais extratos de manjerona e de alecrim apresentaram fraca atividade anti-Sporothrix spp. sobre 40% a 5% dos isolados, não havendo atividade antifúngica sobre as infusões de alecrim preparadas em 10 e 60 minutos. Sobre itraconazol, Sporothrix spp. foi sensível na fase leveduriforme (16 a ≤0.03 μg/mL) e filamentosa (64 a ≤0.12 μg/mL), entretanto, 5 foi observada resistência antifúngica em 4% e 28% (CIM e CFM >16 μg/mL, respectivamente) dos isolados na fase leveduriforme, ao passo que, na fase filamentosa, a resistência ocorreu em 12,5% e 85% dos isolados (CIM e CFM >64 μg/mL, respectivamente). Os óleos essenciais de orégano e manjerona apresentaram maior atividade citotóxica com 80% de inviabilidade celular, ao passo que os extratos aquosos e hidroalcoólico de manjerona foram os menos citotóxicos. A análise química foi similar nos produtos das plantas Lamiaceae, diferindo a concentração dos compostos, os quais α-pineno e 1,8-cineol foram majoritários para o óleo extraído do alecrim e α- terpineno, terpineol-4 e timol para o óleo extraído de orégano, ao passo que 1,8-cineol foi majoritário para os produtos comerciais de alecrim e manjerona, e carvacrol para orégano. A boa atividade anti-Sporothrix spp. in vitro das plantas da família Lamiaceae, em especial ao Origanum vulgare L., é promissora para o tratamento da esporotricose, inclusive sobre isolados clínicos resistentes. / Therapeutics properties of medicinal plants are increasingly studied, mainly due to increasing cases of antimicrobial cases, as observed in strains of Sporothrix Complex. Plants of Lamiaceae family are known for their antifungal properties, but studies in causative agents of sporotrichosis are scarces. Due to the promising potential of these plants, aimed to (1) evaluate the in vitro anti-Sporothrix spp. activity of aqueous extracts of infusion and decoction, hydroalcoholic extract and essential oils os Origanum vulgare L. (oregano), Origanum majorana L. (marjoram) and Rosmarinus officinalis L. (rosemary); (2) evaluate the main chemical constituents present in essential oils; and (3) evaluate the in vitro cytotoxic activity. Aqueous and hydroalcoholic extracts were prepared from the aerial parts of the plants. In essential oils, commercial products and extracted products from aerial parts through hydrodistillation in Clevenger were tested. Both essential oils were chemically analyzed by gas chromatography. In vitro tests were performed by broth microdilution technique against clinical isolates of Sporothrix spp. from humans, dogs and cats with sporotrichosis, as well as environmental soil, in yeast (CLSI-M27A3) and mycelial (CLSI-M38A2) phases and tested from 72 to 0.07 mg/mL. Cytotoxic effects were assessed by MTT assay on VERO cells (78 to 5000 μg/mL). In yeast phase, the extracted and commercial essential oils of oregano showed anti-Sporothrix spp. activity in the minimum inhibitory concentrations (MIC) and minimum fungicidal concentration (MFC) of 36 to ≤2.25 mg/mL and rosemary between 72 to ≤2.25 mg/mL, with no statistical difference between the types of oils. In turn, commercial marjoram oil showed MIC/MFC of ≤2.25 to 18 mg/mL. In the mycelial phase, 100% of feline and canine isolates were sensibles to essential oils of oregano (0.14 to ≤0.07 mg/mL), rosemary (18 to ≤0,07 mg/mL) and marjoram (4.5 to ≤0.07 mg/ml). Aqueous and hydroalcoholic extracts of oregano showed inhibitory and fungicidal activity, respectively, 100% and between 90% and 35% of animal isolates (40 to ≤0.07 mg/mL). In the same concentrations, hydroalcoholic extract of marjoram inhibited 90% to 100% of the isolates. However, other extracts of marjoram and rosemary showed weak activity anti- Sporothrix spp. about 40% to 5% of the isolates, with no antifungal activity by INF10 and INF60 rosemary. About itraconazole, Sporothrix spp. was sensitive in the yeast phase (16 to ≤0.03 mg/mL) and filamentous (64 to ≤0.12 mg/mL), however, antifungal resistance was observed in 4% and 28% (MIC and MFC > 16 μg/mL, respectively) in fungal isolates in the yeast form, and in the mycelial form, the resistence occurred in 12.5% and 85% of isolates (MIC and MFC > 64 μg/mL, respectively). Essential oils of oregano and marjoram exhibited greater cytotoxic activity with 80% cell inviability, while hydroalcoholic and aqueous extracts of marjoram were less cytotoxic. The chemical analysis was similar for the products of Lamiaceae plants and differed in the concentration of the compounds, which α-pinene and 1,8-cineole were majoritary for extracted oil of rosemary and α-terpinene, terpineol-4 and thymol for extracted oil of 7 oregano, while 1,8-cineole was majoritary for commercial products of rosemaru and marjoram, and carvacrol for oregano. The good in vitro anti-Sporothrix spp. activity of the plants of Lamiaceae family, mainly to Origanum vulgare L., is promising for the treatment of sporotrichosis, including against clinical isolates resistant.

As bactérias do gênero Salmonella são, atualmente, uma grande preocupação pela alta incidência de casos registrados, e um sério problema de saúde pública, pela severidade do processo infeccioso. Em função disto os protocolos de controle e legislações em vigilância em saúde obrigam a ausência total de Salmonella em alimento. Para garantir a segurança do alimento, uma crescente demanda de produtos naturais vem sendo estudas para controlar os patógenos alimentares. As plantas condimentares historicamente vêm sendo usadas para conferir sensorialidade aos alimentos, porém muitos desses recursos naturais possuem propriedades antimicrobianas. No entanto, pouco se sabe sobre a interação que essas especiarias influem sobre esses patógenos. Este estudo foi dividido em duas partes. Na primeira, através de testes de diluição em sistemas de tubos múltiplos, determinou-se a Intensidade de Atividade e Inibição Bacteriana (IINIB) e a Intensidade de Atividade e Inativação Bacteriana (IINAB) de diferentes extratos de Artemisia dracunculus L. (“estragão”) e Origanum vulgare L. (“orégano”) frente a oito isolados de Salmonella spp. de sete surtos toxinfectivos alimentares ocorridos no Rio Grande do Sul além de Salmonella Enteritidis ATCC (13076). Foram testados também diferentes formas de extração e suas concentrações, bem como o tempo de contato das bactérias ao extrato. A quantidade de polifenois totais foram, relacionadas à forma de extração (etanólica e hidroetanólico) e ao tipo de extrato da planta. Os resultados foram apresentados como variáveis arbitrárias sendo 9 a atividade máxima e 1 a não atividade bacteriana. Entre as Salmonella spp. houve diferença significativa (p >0,05) em IINIB 7,99 mais resistente a 8,28 mais sensível e os resultado em IINAB 6,77 mais resistente 7,22 mais sensível. As bactérias foram significativamente mais sensíveis aos extratos de estragão do que o de orégano da mesma forma a concentração de 25 % foi mais efetiva que as outras assim como o tempo de contato dos extratos com a bactéria resultou em maior atividade no tempo de 144h. Quanto ao tipo de extração a forma etanólica recebeu maiores notas que a extração Hidroetanólica e quando relacionada a quantidade de fenóis totais foram mais elevadas na etanólica e a planta com maior quantidade de polifenois foi o estragão. Com esses dados pode se dizer que as duas plantas mostraram ação bactericida e bacteriostática frente às salmonelas. Na segunda parte do trabalho contaminou-se carne bovina moída com inóculo padrão final em 104 UFC/mL de salmonela em contato com extratos de Artemisia dracunculus à concentração de 40 % e 45%, o extrato etanólico de Origanum vulgare L. Foi testada a sensibilidade do teste diagnostico de identificação de salmonelas criando uma modificação à técnica padrão oficial do Ministério da Agricultura, Pecuária e Abastecimento (MAPA) acrescentando desestressores que serviram de comparação (teste ouro). Para extrato de estragão observou-se uma sensibilidade do teste em 8 horas de 73,3% e em 24horas de 40% em quanto para orégano a sensibilidade do teste em 8 horas foi de 93,3% e em 24 horas de 6,6 %. Dessa forma o objetivo desse estudo foi trazer à discussão a necessidade de avaliar os processos de ativação das salmonelas originadas de alimentos contaminados, possibilitando uma qualificação dos resultados preditivos do teste oficial. / The Salmonella genus bacteria are currently a major concern because of the high incidence of reported cases, and a serious public health problem, due to the severity of the infectious process. Because of that, the control protocols and laws on health surveillance require a total absence of Salmonella in food. To ensure food security, there is a growing demand for natural products able to control food pathogens. The condiment plants have been historically used to flavor food, but many of these natural resources have antimicrobial properties. However, little is known about the action of the spices on those pathogens. This study was divided in two parts: The first, through dilution tests in a multiple tube testing system, we determined the Intensity of Activity and Bacterial Inhibition (IINIB) and Intensity of Activity and Bacterial Inactivation (IINAB) of different extracts of Artemisia dracunculus L. ("tarragon") and Origanum vulgare L. ("oregano"), compared to eight isolated types of Salmonella spp. from seven alimentary toxinfection outbreaks in Rio Grande do Sul, as well as Salmonella Enteritidis ATCC (13076). Different forms of extraction and their concentrations, as well as the contact time of the bacteria to the extracts were tested. The amount of total polyphenols were related to the form of extraction (ethanolic and hydroethanolic) and the type of the plant extract. The results were presented as arbitrary variables, showing 9 to the maximum activity and 1 to non-bacterial activity. Among Salmonella spp., there was significant difference (p> 0.05) in IINIB, 7.99 the most resistant to 8.28 the most sensitive, and results in IINAB 6.77the most resistant and 7.22 the most sensitive. The bacteria were remarkably more sensitive to tarragon extracts than oregano extracts. Similarly, the 25% concentration was more effective than the others, as well as the contact time of the extracts with the bacteria resulted in a higher activity within 144 h time. Regarding to the type of extraction, the ethanolic form showed higher scores than the hydroethanolic, and when related to the total amount phenols, these were higher in ethanolic extraction, and the highest amount of polyphenols was found in tarragon. With these data we can say that the two plants showed bactericidal and bacteriostatic action on the Salmonella. These data show that the two studied plants showed bactericidal and bacteriostatic action on the Salmonella. The second part of the study consisted in contacting contaminated ground meat with final standard inoculum 104 CFU / ml of Salmonella with extracts of Artemisia dracunculus in 40% and 45% concentration, and ethanol extract of Origanum vulgare. The responsiveness of the diagnosing test for identification of Salmonella was tested, creating a technical amendment to the official standard of the Ministério da Agricultura, Pecuária e Abastecimento (MAPA), adding de-stressors used as a comparison (gold standard). The tarragon extract showed responsiveness to the test within 8 hours at 73.3% and 24 hours at 40%, while for oregano the test responsiveness was within 8 hours at 93.3% and 24 hours at 6.6%. Therefore, the aim of this study was to discuss the need of evaluating the activation processes of the Salmonella originated from contaminated food, enabling a predictive qualification of the official test results.

The objective of that work was to evaluate the effects of light, of temperature and of hydric stress in the germination and vitality of seeds of anise, fennel and dill. The experiment was led in two stages. In stage I, the seeds were placed to germinate to constant temperatures of 20, 25, 30 ºC and alternate temperatures of 20-30 ºC in the presence and absence of light. The assessed parameters were: germination percentage, first count, index of germination speed, length and dry mass of seedlings. In stage II, the seeds were placed in soaked substratum in polyethylene glicol (PEG 6000) in the osmotic potentials equivalent to zero; -0,05; -0,10; -0,15; - 0,20; -0,25; -0,30 MPa. Germination percentage, first count and index of germination speed were evaluated. In stage I, the procedure employed was reached by chance, with the data submitted to variance analysis and to the Tukey test to a 5% of probability, and in stage II, analysis of polinomial regression was used. According to the results, it is possible to conclude that germination of seeds anise, fennel and dill happen in presence and absence light, however the manifestation of the vigor is favored by light. The germination of dill seeds is larger in light presence. The seeds anise, fennel and dill, without dormancy, germinate better in the constant temperatures of 20 and 25 °C and temperature of 30 °C is not adapted for germination test in these species. The dill seeds are more tolerant to hydric stress than the anise and fennel seeds. The decrease of the osmotic potentials reduces the germination and vigor of seeds anise, fennel and dill, being the most affected vigor than germination. The germination is reduced drastically, starting from -0,1 MPa for fennel and -0,2 MPa for dill, when there is decrease of osmotic potentials with polyethylene glicol 6000. / O objetivo desse trabalho foi avaliar os efeitos da luz, da temperatura e do estresse hídrico na germinação e no vigor de sementes de anis, funcho e endro. O experimento foi conduzido em duas etapas. Na etapa I, as sementes foram colocadas para germinar nas temperaturas constantes de 20, 25, 30 ºC e alternada de 20-30 ºC na presença e ausência de luz. Os parâmetros avaliados foram: percentagem de germinação, primeira contagem, índice de velocidade de germinação, comprimento e massa seca das plântulas. Na etapa II, as sementes foram colocadas em substrato embebido em solução de polietileno glicol (PEG 6000) nos potenciais osmóticos correspondentes a zero; -0,05; -0,10; -0,15; -0,20; -0,25; -0,30 MPa. Avaliou-se a percentagem de germinação, primeira contagem e índice de velocidade de germinação. O delineamento utilizado foi o inteiramente casualizado com os dados submetidos à análise de variância e teste de Tukey a 5% de probabilidade, na etapa I e análise de regressão polinomial, na etapa II. Conforme os resultados pode-se concluir que a germinação das sementes de anis, funcho e endro ocorre tanto na presença quanto na ausência de luz, porém a manifestação do vigor é favorecida pela luz. A germinação das sementes de endro é maior na presença de luz. As sementes de anis, funcho e endro, sem dormência, germinam melhor nas temperaturas constantes de 20 e 25 °C e a temperatura de 30 °C não é adequada para o teste de germinação nestas espécies. As sementes de endro são mais tolerantes ao estresse hídrico que as sementes de anis e funcho. A diminuição dos potenciais osmóticos reduz a germinação e o vigor das sementes de anis, funcho e endro, sendo o vigor mais afetado que a germinação. A germinação é reduzida drasticamente, a partir de -0,1 MPa para funcho e -0,2 MPa para endro, quando há diminuição dos potenciais osmóticos com polietileno glicol 6000.

Les plantes produisent une grande diversité de produits naturels parmi lesquels les isoprénoïdes prédominent. Ces molécules ont des fonctions essentielles pour la croissance et le développement : hormones végétales, régulateurs de croissance comme les brassinostéroïdes, pigments photosynthétiques, tous agissant sur des processus biologiques majeurs. Ainsi, la germination, la floraison, la tolérance aux stress thermiques et hydriques, ou la production de semences, sont contrôlées par l’action d’isoprénoïdes. Le but de mon projet est d’identifier par une sélection génétique les éléments régulateurs de l’homéostasie des isoprénoïdes. Pour cela, j’ai réalisé le criblage de suppresseurs de défaut de croissance dans deux mutants de biosynthèse d’isoprénoïdes chez Arabidopsis thaliana et un mutant de signalisation de brassinostéroïdes chez Hordeum vulgare. J’ai par ailleurs étudié la voie de biosynthèse de stérols spécifique ayant lieu dans un type cellulaire particulier, le tube pollinique en croissance. / Plants produce a great diversity of natural compounds, among which isoprenoids prevail. These molecules have essential functions for growth and development: plant hormones, growth regulators as brassinosteroids, photosynthetic pigments, acting on major biological processes. Thus, germination, flowering, heat and draught stress tolerance, or seed production are controlled by the action of isoprenoids. The aim of my project is to identify by genetic selection the regulators of isoprenoid homeostasis. For this, I carried out genetic screens for suppressors of growth defects in two isoprenoid biosynthesis deficient Arabidopsis thaliana mutants and a brassinosteroid signaling Hordeum vulgare mutant. The second part of my project was focused on the specific sterol biosynthetic pathway occurring in a specialized cell type, the germinating pollen tube.