Este trabalho de Mestrado teve por objetivo avaliar a atividade antifúngica in vitro e em queijo Minas Padrão de duas formulações (A e B) de nanoemulsões encapsulando óleo essencial de orégano (Origanum vulgare) produzidas pelo método da temperatura de inversão de fases (método PIT). Cladosporium sp., Fusarium sp. e Penicillium sp. foram os fungos identificados nas amostras de queijo Minas Padrão deteriorados. Tais cepas foram utilizadas na avaliação da atividade antifúngica in vitro e em queijo Minas Padrão do óleo de orégano nanoemulsionado. Nos testes in vitro foram determinadas as concentrações mínimas inibitórias (CMI) do óleo essencial de orégano puro e nanoemulsionado sobre o Cladosporium sp., Fusarium sp. e Penicillium sp. Foram utilizadas fatias de queijo Minas Padrão para avaliar o efeito inibitório das nanoemulsões contendo óleo essencial de orégano. Por último foi avaliado o efeito inibitório das nanoemulsões durante o processo de maturação de queijos Minas Padrão. Determinaram-se valores de CMI para óleo essencial puro de 0,2; 0,3 µg/ml para Fusarium sp. e Penicillium sp., respectivamente. Enquanto para as duas formulações de nanoemulsão; os valores de CMI dependeram da quantidade de óleo essencial de orégano que estava contido nelas, obtendo-se valores de 0,26; 0,11 e 1,62 µg/ml para a formulação A de óleo de orégano nanoemulsionado sobre Cladosporium sp., Fusarium sp. e Penicillium sp., respectivamente, e CMIs de 0,32; 0,1 e 0,8 µg/ml para a formulação B de nanoemulsão sobre os mesmos gêneros de fungos. Os ensaios nas fatias de queijo evidenciaram que o efeito das CMIs, quando aplicadas na matriz alimentícia, foi nulo, permitindo o desenvolvimento normal dos fungos avaliados, da mesma forma se determinou a importância da atividade de água no crescimento fúngico. Já nos queijos em ambiente de maturação o efeito inibitório do óleo de orégano foi pouco satisfatório, o que indicou a importância do controle dos demais parâmetros ambientais no ambiente de maturação. Pode-se concluir que o óleo essencial de orégano nanoemulsionado apresentou efeito inibitório contra os gêneros de fungos avaliados. Quando controlados parâmetros ambientais como temperatura de armazenamento e atividade de água, seu efeito inibitório pode ser amplamente melhorado, apresentando-o como potencial alternativa na conservação dos alimentos. / This Master Thesis aimed to evaluate the in vitro antifungal activity and in Minas Padrão cheese of two formulations (A and B) of nanoemulsions encapsulating essential oil of oregano (Origanum vulgare) produced by the method of phase inversion temperature (PIT method). Cladosporium sp., Fusarium sp. and Penicillium sp. fungi were identified in deteriorated cheese samples. These strains were used in the evaluation of antifungal activity of oregano essential oil nanoemulsions, both in vitro and in cheese. Minimum inhibitory concentrations (MIC) of essential oil of pure oregano oil and nanoemulsions were determined. Cheese slices were used to evaluate the inhibitory effect of nanoemulsions containing essential oil of oregano. Finally the inhibitory effect of encapsulated oregano oil was tested during cheese ripening. MIC values determined for pure essential oil were 0.20 and 0.3 µg/ml for Fusarium sp. and Penicillium sp., respectively. As for the two nanoemulsion formulations, MIC values depended on the amount of essential oil of oregano which was contained therein, and the values for formulation A were 0.26, 0.11 and 1.62 µg/ml for Cladosporium sp., Fusarium sp. and Penicillium sp, respectively. For formulation B, the values of MIC were 0.32, 0.10 and 0.80 µg/ml. The tests on the cheese slices showed that the effect of MIC when applied in the food matrix was null, as the fungi growth was not avoided. As for the tests during cheese ripening, the inhibitory effect of oregano oil in nanoemulsions was unsatisfactory, which indicated the importance of controlling other environmental parameters in the maturation chamber. It can be concluded that the nanoemulsions of essential oil presented inhibitory effect against the genera of fungi evaluated. Controlled environmental parameters such as storage temperature and water activity, its inhibitory effect can be highly improved, and the nanoemulsions of oregano oil can be seen as a potential system for food preservation.

Este trabalho de Mestrado teve por objetivo avaliar a atividade antifúngica in vitro e em queijo Minas Padrão de duas formulações (A e B) de nanoemulsões encapsulando óleo essencial de orégano (Origanum vulgare) produzidas pelo método da temperatura de inversão de fases (método PIT). Cladosporium sp., Fusarium sp. e Penicillium sp. foram os fungos identificados nas amostras de queijo Minas Padrão deteriorados. Tais cepas foram utilizadas na avaliação da atividade antifúngica in vitro e em queijo Minas Padrão do óleo de orégano nanoemulsionado. Nos testes in vitro foram determinadas as concentrações mínimas inibitórias (CMI) do óleo essencial de orégano puro e nanoemulsionado sobre o Cladosporium sp., Fusarium sp. e Penicillium sp. Foram utilizadas fatias de queijo Minas Padrão para avaliar o efeito inibitório das nanoemulsões contendo óleo essencial de orégano. Por último foi avaliado o efeito inibitório das nanoemulsões durante o processo de maturação de queijos Minas Padrão. Determinaram-se valores de CMI para óleo essencial puro de 0,2; 0,3 µg/ml para Fusarium sp. e Penicillium sp., respectivamente. Enquanto para as duas formulações de nanoemulsão; os valores de CMI dependeram da quantidade de óleo essencial de orégano que estava contido nelas, obtendo-se valores de 0,26; 0,11 e 1,62 µg/ml para a formulação A de óleo de orégano nanoemulsionado sobre Cladosporium sp., Fusarium sp. e Penicillium sp., respectivamente, e CMIs de 0,32; 0,1 e 0,8 µg/ml para a formulação B de nanoemulsão sobre os mesmos gêneros de fungos. Os ensaios nas fatias de queijo evidenciaram que o efeito das CMIs, quando aplicadas na matriz alimentícia, foi nulo, permitindo o desenvolvimento normal dos fungos avaliados, da mesma forma se determinou a importância da atividade de água no crescimento fúngico. Já nos queijos em ambiente de maturação o efeito inibitório do óleo de orégano foi pouco satisfatório, o que indicou a importância do controle dos demais parâmetros ambientais no ambiente de maturação. Pode-se concluir que o óleo essencial de orégano nanoemulsionado apresentou efeito inibitório contra os gêneros de fungos avaliados. Quando controlados parâmetros ambientais como temperatura de armazenamento e atividade de água, seu efeito inibitório pode ser amplamente melhorado, apresentando-o como potencial alternativa na conservação dos alimentos. / This Master Thesis aimed to evaluate the in vitro antifungal activity and in Minas Padrão cheese of two formulations (A and B) of nanoemulsions encapsulating essential oil of oregano (Origanum vulgare) produced by the method of phase inversion temperature (PIT method). Cladosporium sp., Fusarium sp. and Penicillium sp. fungi were identified in deteriorated cheese samples. These strains were used in the evaluation of antifungal activity of oregano essential oil nanoemulsions, both in vitro and in cheese. Minimum inhibitory concentrations (MIC) of essential oil of pure oregano oil and nanoemulsions were determined. Cheese slices were used to evaluate the inhibitory effect of nanoemulsions containing essential oil of oregano. Finally the inhibitory effect of encapsulated oregano oil was tested during cheese ripening. MIC values determined for pure essential oil were 0.20 and 0.3 µg/ml for Fusarium sp. and Penicillium sp., respectively. As for the two nanoemulsion formulations, MIC values depended on the amount of essential oil of oregano which was contained therein, and the values for formulation A were 0.26, 0.11 and 1.62 µg/ml for Cladosporium sp., Fusarium sp. and Penicillium sp, respectively. For formulation B, the values of MIC were 0.32, 0.10 and 0.80 µg/ml. The tests on the cheese slices showed that the effect of MIC when applied in the food matrix was null, as the fungi growth was not avoided. As for the tests during cheese ripening, the inhibitory effect of oregano oil in nanoemulsions was unsatisfactory, which indicated the importance of controlling other environmental parameters in the maturation chamber. It can be concluded that the nanoemulsions of essential oil presented inhibitory effect against the genera of fungi evaluated. Controlled environmental parameters such as storage temperature and water activity, its inhibitory effect can be highly improved, and the nanoemulsions of oregano oil can be seen as a potential system for food preservation.

Atualmente, a população mundial está passando por um processo de transição demográfica, relacionado com o aumento considerável do número de idosos em relação aos jovens. O rápido crescimento da população idosa constitui uma das maiores preocupações da sociedade moderna, o que demanda um aumento no estudo da saúde e da melhoria da qualidade de vida dos idosos, incluindo um maior esclarecimento dos aspectos patofisiológicos relacionados com as doenças microbianas. Uma das infecções que afetam os idosos, principalmente os imunocomprometidos, é a candidose, a qual pode ser causada por diferentes espécies de Candida, em especial Candida albicans (C. albicans). Os monócitos e macrófagos exercem funções essenciais no combate a microrganismos através do processo fagocítico que abrange a produção de espécies reativas do nitrogênio e oxigênio que culminam no controle e/ou morte do patógeno. Uma vez esses fatores comprometidos, o combate aos agentes agressores é dificultado e ou ineficaz fazendo com que estas disfunções ocasionem problemas clínicos. Sendo assim, o objetivo do presente trabalho foi avaliar in vitro a capacidade fagocítica, a geração de óxido nítrico (NO) e peróxido de hidrogênio (H2O2) intracelular, e a habilidade de controlar o crescimento extracelular do fungo C. albicans por monócitos e macrófagos humanos de idosos e jovens. Para isso, foram obtidos monócitos do sangue periférico de indivíduos idosos e jovens os quais permaneceram em cultura por 7 dias e se diferenciaram em macrófagos. Ambos os fagócitos foram desafiadas com diferentes proporções de C. albicans viáveis (5 células:1 fungo, 1:1 e 1:5) por 30min, 2h e 5h. Após os desafios, os dados foram analisados por meio de ensaio fluorescente de fagocitose e crescimento fúngico extracelular, e pelos kits DAF-FM diacetato (NO) e Cell Rox Deep (H2O2). Os resultados foram expressos como média ± desvio padrão (SD) dos valores ou porcentagens obtidos e a análise estatística foi realizada por meio do teste estatístico ANOVA fatorial seguido do teste de Fischer, considerando p<0,05. Nossos resultados revelaram que a idade não afeta a internalização de C. albicans por monócitos e macrófagos oriundos de idosos, no entanto, esses indivíduos levaram mais tempo para ativar a produção de NO e H2O2 que os jovens. Após 5h de estímulo fúngico, as células dos idosos se igualaram ou superaram a resposta imunológica apresentada pelos jovens, demonstrando maior habilidade em controlar o crescimento fúngico extracelular. Baseados nestes resultados, concluímos que, em comparação aos jovens, os monócitos e macrófagos de idosos têm sua habilidade de internalizar o fungo C. albicans preservada; no entanto, os seus sinais de ativação para a produção de substâncias microbicidas, como NO e H2O2, estão prejudicados, o que pode propiciar a instalação local do fungo e/ou disseminação sistêmica nesta população. Além disso, ao longo do tempo, estas células oriundas de idosos conseguiram produzir NO e H2O2 como ou mais que os jovens, sugerindo um estado de pré-ativação celular em função de níveis elevados de citocinas pró-inflamatórias inerentes do estado crônico inflamm-aging, o qual os idosos podem estar vivenciando. / Currently, the world population is undergoing a process of demographic transition, associated with a considerable increase in the number of elderly compared with young subjects. The rapid growth of the elderly population is a major concern of the modern society, which requires an increase in the study of health and improved quality of life for the aged. This includes a greater clarification of the pathophysiological aspects of microbial diseases. One of the infections affecting the elderly, particularly those immunocompromised, is the candidosis that can be caused by various Candida species, especially Candida albicans (C. albicans). Monocytes and macrophages exert essential functions in combating microorganisms by phagocytic process. During this process occur the production of reactive nitrogen species and reactive oxygen species that culminate in the control and/or death of the pathogen. Once these factors are affected, the fight against aggressors is difficult and/or ineffective, and these disorders can cause clinical problems. Thus, the aim of this in vitro study was to investigate occurrence of phagocytosis, the intracellular generation of nitric oxide (NO) and hydrogen peroxide (H2O2), and the ability of human monocytes and macrophages from young and old in controlling the extracellular growth of the fungus C. albicans. Therefore, peripheral blood monocytes from elderly and young subjects were obtained and remained in culture for 7 days until they differentiate into macrophages. Both mononuclear cells were challenged with different proportions of viable C. albicans (5 cells:1 fungus, 1:1 and 1:5) for 30min, 2h and 5h. After the challenges, it was possible to evaluate the ability of fungal internalization and the controlling of extracellular fungal growth by a fluorescent assay, and measure the intracellular production of nitric oxide (NO) by DAF-FM Diacetate and hydrogen peroxide (H2O2) by Rox Deep Cell Kit. The results were expressed as mean ± standard deviation (SD) values or percentages obtained and the statistical analysis was performed using factorial ANOVA followed by Fisher test, with p <0.05. Our results showed that age does not affect the internalization of C. albicans by monocytes and macrophages derived from older subjects, however, these individuals took more time to activate the production of NO and H2O2 than that young. After 5h of fungal stimulation, the cells of the elderly equaled or exceeded the immune response presented by young people, demonstrating greater ability to control the extracellular fungal growth. Thus, we conclude that, compared with young, monocytes and macrophages from elderly have their ability to internalize the fungus C. albicans preserved; however, their activation signals for the production of microbicidal substances, such as NO and H2O2 are impaired, which may facilitate the local installation of the fungus and/or systemic dissemination in this population. Furthermore, over time, these cells derived from elderly were able to produce NO and H2O2 as or more than the young, suggesting a state of cellular pre-activation. Probably due to high levels of proinflammatory cytokines involved in the chronic state which older people may be experiencing, known as inflamm-aging.

Atualmente, a população mundial está passando por um processo de transição demográfica, relacionado com o aumento considerável do número de idosos em relação aos jovens. O rápido crescimento da população idosa constitui uma das maiores preocupações da sociedade moderna, o que demanda um aumento no estudo da saúde e da melhoria da qualidade de vida dos idosos, incluindo um maior esclarecimento dos aspectos patofisiológicos relacionados com as doenças microbianas. Uma das infecções que afetam os idosos, principalmente os imunocomprometidos, é a candidose, a qual pode ser causada por diferentes espécies de Candida, em especial Candida albicans (C. albicans). Os monócitos e macrófagos exercem funções essenciais no combate a microrganismos através do processo fagocítico que abrange a produção de espécies reativas do nitrogênio e oxigênio que culminam no controle e/ou morte do patógeno. Uma vez esses fatores comprometidos, o combate aos agentes agressores é dificultado e ou ineficaz fazendo com que estas disfunções ocasionem problemas clínicos. Sendo assim, o objetivo do presente trabalho foi avaliar in vitro a capacidade fagocítica, a geração de óxido nítrico (NO) e peróxido de hidrogênio (H2O2) intracelular, e a habilidade de controlar o crescimento extracelular do fungo C. albicans por monócitos e macrófagos humanos de idosos e jovens. Para isso, foram obtidos monócitos do sangue periférico de indivíduos idosos e jovens os quais permaneceram em cultura por 7 dias e se diferenciaram em macrófagos. Ambos os fagócitos foram desafiadas com diferentes proporções de C. albicans viáveis (5 células:1 fungo, 1:1 e 1:5) por 30min, 2h e 5h. Após os desafios, os dados foram analisados por meio de ensaio fluorescente de fagocitose e crescimento fúngico extracelular, e pelos kits DAF-FM diacetato (NO) e Cell Rox Deep (H2O2). Os resultados foram expressos como média ± desvio padrão (SD) dos valores ou porcentagens obtidos e a análise estatística foi realizada por meio do teste estatístico ANOVA fatorial seguido do teste de Fischer, considerando p<0,05. Nossos resultados revelaram que a idade não afeta a internalização de C. albicans por monócitos e macrófagos oriundos de idosos, no entanto, esses indivíduos levaram mais tempo para ativar a produção de NO e H2O2 que os jovens. Após 5h de estímulo fúngico, as células dos idosos se igualaram ou superaram a resposta imunológica apresentada pelos jovens, demonstrando maior habilidade em controlar o crescimento fúngico extracelular. Baseados nestes resultados, concluímos que, em comparação aos jovens, os monócitos e macrófagos de idosos têm sua habilidade de internalizar o fungo C. albicans preservada; no entanto, os seus sinais de ativação para a produção de substâncias microbicidas, como NO e H2O2, estão prejudicados, o que pode propiciar a instalação local do fungo e/ou disseminação sistêmica nesta população. Além disso, ao longo do tempo, estas células oriundas de idosos conseguiram produzir NO e H2O2 como ou mais que os jovens, sugerindo um estado de pré-ativação celular em função de níveis elevados de citocinas pró-inflamatórias inerentes do estado crônico inflamm-aging, o qual os idosos podem estar vivenciando. / Currently, the world population is undergoing a process of demographic transition, associated with a considerable increase in the number of elderly compared with young subjects. The rapid growth of the elderly population is a major concern of the modern society, which requires an increase in the study of health and improved quality of life for the aged. This includes a greater clarification of the pathophysiological aspects of microbial diseases. One of the infections affecting the elderly, particularly those immunocompromised, is the candidosis that can be caused by various Candida species, especially Candida albicans (C. albicans). Monocytes and macrophages exert essential functions in combating microorganisms by phagocytic process. During this process occur the production of reactive nitrogen species and reactive oxygen species that culminate in the control and/or death of the pathogen. Once these factors are affected, the fight against aggressors is difficult and/or ineffective, and these disorders can cause clinical problems. Thus, the aim of this in vitro study was to investigate occurrence of phagocytosis, the intracellular generation of nitric oxide (NO) and hydrogen peroxide (H2O2), and the ability of human monocytes and macrophages from young and old in controlling the extracellular growth of the fungus C. albicans. Therefore, peripheral blood monocytes from elderly and young subjects were obtained and remained in culture for 7 days until they differentiate into macrophages. Both mononuclear cells were challenged with different proportions of viable C. albicans (5 cells:1 fungus, 1:1 and 1:5) for 30min, 2h and 5h. After the challenges, it was possible to evaluate the ability of fungal internalization and the controlling of extracellular fungal growth by a fluorescent assay, and measure the intracellular production of nitric oxide (NO) by DAF-FM Diacetate and hydrogen peroxide (H2O2) by Rox Deep Cell Kit. The results were expressed as mean ± standard deviation (SD) values or percentages obtained and the statistical analysis was performed using factorial ANOVA followed by Fisher test, with p <0.05. Our results showed that age does not affect the internalization of C. albicans by monocytes and macrophages derived from older subjects, however, these individuals took more time to activate the production of NO and H2O2 than that young. After 5h of fungal stimulation, the cells of the elderly equaled or exceeded the immune response presented by young people, demonstrating greater ability to control the extracellular fungal growth. Thus, we conclude that, compared with young, monocytes and macrophages from elderly have their ability to internalize the fungus C. albicans preserved; however, their activation signals for the production of microbicidal substances, such as NO and H2O2 are impaired, which may facilitate the local installation of the fungus and/or systemic dissemination in this population. Furthermore, over time, these cells derived from elderly were able to produce NO and H2O2 as or more than the young, suggesting a state of cellular pre-activation. Probably due to high levels of proinflammatory cytokines involved in the chronic state which older people may be experiencing, known as inflamm-aging.

Sammanfattning: Wood decay fungi are the main decomposer of lignocellulose material stored in wood. Thus, all factors that affect them could affect their ecological function. This in return, may affect ecosystem functioning in terms of altered carbon emissions from dead wood. Increased temperature is one of the main factors influencing fungal decay. The aim of the current study is to explore the effects of temperature and assembly history (order of species arrival), two important regulators of fungal communities, on wood decomposition. I conducted a microcosm experiment with two temperature treatments and eight assembly histories where each species was allowed to colonize the wood two weeks ahead of the rest of the species. The temperature treatments were set to mimic the effect of climate induced warming. Therefore, I had one treatment with relatively high temperature, representing the expected temperatures year 2100 given the current emission trends of the northern inland of Sweden, and another treatment representing the current normal temperature (1961-1990). The temperature treatments had an average difference of 5°C. In order to see how climate induced warming and fungal assembly history influenced decomposition, I measured and analyzed initial fungal growth, fungal respiration and wood weight loss. Both temperature and assembly history had a significant influence on fungal growth, fungal respiration and wood decomposition. There was also strong interaction between the two factors. The average increase in mass loss under elevated temperature was 19% compared to 14% under normal temperature. The highest mass loss (25%) was when Phlebia centrifuga was the initial species under elevated temperature and the lowest (12%) was when Climacocystis borealis was initial species under normal temperature. All assembly histories had higher mass loss under elevated temperature, but the magnitude varied. For example, when C. borealis was the initial species, mass loss increased by 60% compared to only 7% when Antrodia sinuosa was the initial species. Six out of eight assembly histories had higher CO2 under elevated temperature, with the highest increase (88%) in P. centrifuga histories and the lowest (7%) in C. borealis histories. Even if the results need to be confirmed by field studies, my data illustrates that climate induced warming probably results in higher fungal respiration and deadwood decomposition and that the magnitude of this effect depends on fungal assembly history.

Background – Feeding horses round bale hay (RBH) has been associated with airway inflammation. The purpose of this study was to determine if horses fed RBH for a 6-week period demonstrated more evidence of airway inflammation than horses fed square bale hay (SBH) of comparable quality. Hypothesis - The respiratory health of horses fed RBH will not differ from horses fed SBH of comparable quality. Animals – Two feeding groups of 15 healthy horses (mixed ages, breeds) from the University riding program. Methods – This was a prospective study performed during fall of 2009. At the beginning and end of a 6- week feeding trial, horses were examined (physical, upper airway endoscopic) and samples (tracheal aspirate (TA), bronchoalveolar lavage (BAL)) collected for cytology and/or bacterial/fungal culture. Hay was analyzed for nutritional value and bacterial/fungal content. Results – Horses fed RBH demonstrated an increase in pharyngeal lymphoid hyperplasia (p=0.0143) and percentage neutrophils (p=0.0078) in the TA samples post-feeding as compared to pre-feeding values. Nutritional analysis of hay and measurements of bacterial/fungal load did not differ over time and/or between hay types. Conclusions and clinical importance – The identification of airway inflammation in the horses fed RBH indicates that factors associated with the manner in which the hay is fed and consumed contribute to the development of subclinical airway inflammation. RBH affords horses continuous daily exposure to hay and as horses bury their muzzles in the bale, exposure to particulate matter is likely increased. These factors may partially explain the response in horses fed RBH. Further studies are required to confirm these predictions. / Master of Science