O exossomo é um complexo constituído por até 11 subunidades (Rrp4p, Rrp6p, Rrp40p, Rrp41p, Rrp42p, Rrp43p, Rrp44p, Rrp45p, Rrp46p, Csl4p, Mtr3p) que possui atividade exorribonucleolítica 3`→5` e está envolvido no processamento e degradação de vários tipos de RNAs na célula eucariótica. O complexo tem sido estudado em diversos organismos, como leveduras, insetos, plantas, humanos e também em várias espécies de archaea. Apesar da conservação da estrutura do exossomo ao longo da evolução e de oito subunidades do exossomo eucariótico apresentarem domínios de RNase, apenas duas proteínas, Rrp6p e Rrp44p têm atividade catalítica. A despeito da importância do exossomo para a célula, ainda não está claro o papel de cada subunidade na atividade do complexo. Neste trabalho foram utilizados mutantes da subunidade Rrp43p a fim de avaliar como mutações pontuais nesta subunidade afetam a montagem e estabilização do complexo do exossomo de Saccharomyces cerevisiae. Ensaios de purificação do exossomo com TAP-Rrp43p revelaram que os mutantes co-purificam Mtr3p e Rrp44p menos eficientemente. Além disso, os mutantes também apresentam atividade exorribonucleolítica 3`→5` reduzida, indicando que o defeito na montagem do complexo pode afetar a sua atividade enzimática. / The exosome is a protein complex comprised of up to eleven subunits (Rrp4p, Rrp6p, Rrp40p, Rrp41p, Rrp42p, Rrp43p, Rrp44p, Rrp45p, Rrp46p, Csl4p and Mtr3p) that has 3`→5` exoribonucleolytic activity and is involved in degradation and processing pathways of several kinds of RNA in eukaryotes. This complex has also been identified in several organisms, such as yeast, insects, plants, humans and also many species of archaea. Despite the overall structure conservation of the complex throughout evolution and eight of the eukaryotic exosome subunits displaying RNase domains, only two proteins, Rrp6p and Rrp44p have catalytic activity. Although the exosome has been shown to be involved in many different aspects of RNA metabolism, the role that each subunit plays in the activity of the complex has not yet been determined. In this work we used of TAP-purified exosome complexes to study the effect of Rrp43p mutations on the assembly and stabilization of the complex in Saccharomyces cerevisiae. Co-immunoprecipitation assays revealed that Rrp43p mutants co-purify Mtr3p and Rrp44p subunits less efficiently. Besides, Rrp43p mutants also present decreased activity, indicating that an assembly defect may affect its enzymatic activity

Les levures non-Saccharomyces, naturellement présentes dans les moûts, peuvent impacter positivement ou négativement la qualité des vins. Depuis quelques années, l’utilisation de cultures mixtes comme starters, associant une souche de Saccharomyces cerevisiae et une souche d’une autre espèce est proposée aux œnologues. C’est le cas du couple S. cerevisiae/Torulaspora delbrueckii. L’étude des interactions entre la souche T. delbrueckii Zymaflore Alpha et S. cerevisiae Zymaflore X5, de la société Laffort, a été réalisée. Les fermentations alcooliques ont été effectuées dans un réacteur à double compartiment permettant la séparation physique des levures tout en conservant l’homogénéité du milieu de culture. Les résultats ont mis en évidence que la séparation impacte la croissance des deux souches suggérant l’existence d’interactions de type cell-cell contact entre ces deux souches. Si une grande majorité de praticiens utilise désormais les levures sélectionnées, certains ont fait le choix de favoriser les populations autochtones de levures S .cerevisiae et de levures non-Saccharomyces. L’incidence de deux facteurs de l’environnement a été étudié sur un mélange de cinq espèces de non-Saccharomyces (T. delbrueckii, Metschnikowia spp., Candida zemplinina, Hanseniaspora uvarum, Pichia kluyveri) et de deux souches de S. cerevisiae (une à phase de latence courte, une à phase de latence longue) en cultures pures et en mélange. L’inoculation de la souche de S. cerevisiae à phase de latence longue dans un moût saturé en CO2 permet de stimuler les levures non-Saccharomyces d’intérêt (T. delbrueckii/P. kluyveri) tout en inhibant les espèces indésirables (H. uvarum, C. zemplinina). / Non-Saccharomyces yeasts, naturally found in grape must, can impact wine quality positively or negatively. In recent years, the use of mixed cultures as starters (association of S. cerevisiae species and other species) such as the couple Saccharomyces cerevisiae/Torulaspora delbrueckii is proposed to winemakers. Interactions between these two species have been studied with two commercial strains, T. delbrueckii Zymaflore Alpha and S. cerevisiae Zymaflore X5 (Laffort). Alcoholic fermentations were carried out in a fermentor with double compartment allowing a physical separation of yeasts and preserving the homogeneity culture medium. The results highlighted that the physical separation impacts the growth of both strains, suggesting interactions of type cell-cell contact between these two strains. If a large majority of winemakers use selected yeasts strains, some of them chose to favor native yeasts, S. cerevisiae species and non- Saccharomyces species. The impact of two environmental factors was investigated on five non-Saccharomyces species (T. delbrueckii, Metschnikowia spp., Candida zemplinina, Hanseniaspora uvarum, Pichia kluyveri) and two strains of S. cerevisiae (one with short fermentation lag phase, one with long fermentation lag phase), in pure and mixed cultures. The inoculation with S. cerevisiae with a long fermentation lag phase in a must saturated with CO2 allowed to stimulate some of non-Saccharomyces which present an interest in winemaking (T. delbrueckii/P. kluyveri) and inhibit the undesirable ones (H. uvarum, C. zemplinina).

O objetivo do estudo foi avaliar os efeitos da suplementação de levedura autolisada de Saccharomyces cerevisiae fornecida em duas diferentes inclusões em dietas para frangos de corte como alternativa a um antimicrobiano sobre desempenho zootécnico e avaliação do sistema imune intestinal pela realização da enumeração bacteriana, citometria de fluxo e expressão intestinal de genes ligados à resposta imune intestinal. Neste estudo foram utilizados 1260 pintos de corte machos de um dia de idade da linhagem ROSS AP95&reg; em um experimento de 1 a 35 dias de idade alojados em galpão climatizado com cama de casa de arroz nova. O experimento foi realizado em delineamento inteiramente aleatorizado, com 4 tratamentos e 7 repetições, com 45 aves por boxe. Os tratamentos utilizados foram: T1: ração basal e sem aditivo - controle negativo; T2: ração basal suplementada com 55 ppm de bacitracina de zinco - controle positivo; T3: controle negativo + 2 kg/t de levedura autolisada; T4: controle negativo + 4 kg/t de levedura autolisada. As dietas foram à base de milho e farelo de soja, sendo adicionados às rações 5% de farelo de trigo e 5% de farinha de penas e vísceras (sem tratamento prévio) com objetivo de impor um desafio nutricional aos animais. Ainda visando estimular imunologicamente os animais, aos 7 dias de idade, todas as aves foram desafiadas via ocular com uma vacina viva contendo oocistos de Eimeria sp. na dose recomendada pelo fabricante. Aos 8 dias de idade e 21 dias de idade, uma ave de cada unidade experimental, sem jejum prévio, teve sangue coletado e foi sacrificada para coleta de conteúdo intestinal ileal e cecal para realização da emumeração bacteriana de Enterococus sp., Escherichia coli e Lactobacillus sp., e para a coleta do segmento ileal para avaliar a expressão gênica intestinal de Claudin-1, IL-1&beta;, IL-4, TLR4 e MUC-2 através da PCR em tempo real. Em relação ao desempenho das aves, o tratamento T3 propiciou melhor conversão alimentar em relação a T1 até os 21 dias de idade. Para o período cumulativo, o tratamento T4 propiciou conversão alimentar semelhante ao T2, sendo esta variável melhor para estes tratamentos em relação ao controle negativo. Na enumeração de bactérias no íleo, aos 8 dias de idade, os tratamentos T3 e T4 modularam de forma distinta a contagem de Enterococus sp., e para o gênero Lactobacillus sp., ambos os grupos de levedura apresentaram menor contagem em contraste com o controle positivo. No conteúdo do ceco foi encontrado um menor número de E. coli para os animais grupo T3, diferentemente para o T2 que propiciou maior contagem. Aos 21 dias de idade, foi encontrado diferença na enumeração do gênero Enterococus sp. ileal, cuja contagem foi menor para o T2 em relação ao T1. Na na análise de citometria de fluxo, tendências foram observadas aos 8 dias de idade para o percentual de linfócitos T auxiliares (P=0,16) e para o percentual de linfócitos B (P=0,12) havendo redução com a suplementação de levedura autolisada. A mesma tendência (P=0,19) foi observada aos 21 dias de idade para a contagem de células T citóxicos. Sobre a PCR em tempo real, não foram detectadas diferenças para a expressão de Claudin-1. T2 e T4 propiciaram aumento da expressão gênica de IL-1&beta; aos 21 dias de idade em relação ao controle negativo, sendo que T2 também promoveu aumento de TLR-4 aos 8 dias de idade. Tendências foram observadas com a maior expressão de IL-4 (P=0,06) aos 21 dias de idade pelo T2 e aumento na expressão de MUC-2 (P=0,09) pelo T4 aos 8 dias de idade. Os diferentes padrões de ativação ou não de citocinas revela uma estimulação da via Th2 pelo controle positivo (aumento de IL-1&beta; e IL-4) e da via Th17 pelo tratamento suplementado com 4 kg/t de levedura autolisada (aumento de IL-1&beta;). / The objective of this study was to evaluate the effects of a Saccharomyces cerevisiae autolyzed yeast supplementation in substitution of AGP in broiler diets on performance and immune system (on two different feed inclusions for broilers diets in replacing AGP on broiler performance and evaluation of immune system trough bacterial enumeration, flux citometry and intestinal gene expression. For that, 1260 one-day-old male Ross AP95 chicks were raised from 1 to 35 days of age in a poultry house with new rice husk as litter. The experiment was arranged in a completely randomized design with 4 treatments and 7 replications, with 45 birds per pen. The treatments were: T1: basal diet and no additive - negative control; T2: basal diet supplemented with 55 ppm of zinc bacitracin - positive control; T3: negative control + 2 kg/t of autolyzed yeast; T4: negative control + 4 kg/t of autolyzed yeast. The corn-soybean meal based diets contained 5% wheat bran and 5% poultry by-product meal (with no previous treatment) in order to impose a nutritional challenge to the animals. To impose a further immunological challenge, at 7 days of age, all the birds were eye drop-vaccinated with live vaccine containing Eimeira sp. oocysts at the manufacturer recommended dosis. At 8 and 21 days of age, one chick per experimental unit, with no fasting, had the blood collected and was sacrificed for sampling the ileal and cecal intestinal contents for enumeration of Enterococus sp., Escherichia coli and Lactobacillus sp. Also, the ileal segment was sampled for intestinal gene expression of Claudin-1, IL-1&beta;, IL-4, TLR4 e MUC-2 by RNA extraction through real time PCR. For the performance results at 21 days of age, T3 had the same feed conversion rate of T1. For the cumulative grow-out, T4 had the same feed conversion rate as T2, being this variable better for the aforementioned tretaments in comparison to negative control. For ileal bacterial enumeration, at 8 days of age, T3 and T4 modulated distinctly the enumeration of Enterococus sp., and reduced the counts of Lactobacillus sp. in comparison to the positive control. In the cecal contents, the enumeration for E. coli was the lowest for T3, differing from the positive control. At 21 days of age, there was a difference in ileal Enterococus sp., with higher counts for T2 relative to T1. In the flux citometry, tendencies were observed at 8 days of age for T helper cells (P=0,16) and for B cells (P=0,12), which were reduced in the autolyzed yeast treatments. The same tendency (p=0.19) was seen at 21 days of age for T activated cytotoxic cells. For the real time PCR, there was no difference in the expression of Claudin-1 (P<0,05). T2 and T4 promoted upregulation of IL-1&beta; at 21 days of age (P<0,05) in comparison to the negative control; additionally, the antibiotic tretatment also upregulated the expression of TLR-4 at 8 days of age (P<0,05). Tendencies were observed as upregulation of IL-4 (P=0,06) at 21 days of age by positive control and upregulation of MUC-2 (P=0,09) by the treatment with 4 kg/t of autolyzed yeast at 8 days of age. The different profiles in activating or not cytokines reveals a stimulation of Th2 pathway for the positive control (upregulation of IL-1&beta; and IL-4) and Th17 pathway for the treatment supplemented with 4 kg/t of autolyzed yeast (upregulation of IL-1&beta;).

<p>&Scaron;iroka upotreba imunosupresivnih terapija zajedno sa antimikotičnim terapijama &scaron;itokog spektra značajno je povećala učestalost povr&scaron;inskih i sistemskih infekcija uzrokovanih patogenim kvascem Candida glabrata. Zbog povećane rezistentnosti vrste C. glabrata na klasične antimikotike, javila se potreba za pronalaskom novih alternativa u prevenciji i lečenju infekcija izazvanih ovim patogenom. Tokom poslednjih nekoliko godina se u velikoj meri razmatra potencijalni benefit probiotskih mikroorganizama na zdravlje ljudi. Pored primene brojnih probiotskih bakterija, Saccharomyces boulardii je jedina vrsta kvasca za koju je dokazano da poseduje svojstva koja karakteri&scaron;u probiotike.<br />Cilj istraživanja ove doktorske disertacije je ispitivanje uticaja probiotskog kvasca S. boulardii na adheziju kvasca C. glabrata na polistitren povr&scaron;inu pri različitim temperaturama, pH vrednostima i u prisustvu različitih koncentracija tri klinički važna antimikotika kao &scaron;to su flukonazol, itrakonazol i amfotericin B. Hidrofobnost povr&scaron;ine ćelije (CSH) sojeva kvasca C. glabrata je određena u cilju procene korelacije između ove fizičko-hemijske osobine i adhezije na polistiren povr&scaron;inu. Pored toga, ispitana je adhezija kvasca C. glabrata u ko-kulturi sa drugim vrstama mikroorganizama. Testirana su tri soja kvasca C. glabrata, kvasci Candida krusei i Saccharomyces cerevisiae, kao i dve vrste probiotskih bakterija Lactobacillus rhamnosus i Lactobacillus casei. Metoda kori&scaron;ćena za procenu adhezije bila je bojenje kristal violetom, dok je hidrofobnost povr&scaron;ine ćelije sojeva kvasca C. glabrata određena testom Mikrobne adhezije na ugljovodonike.<br />Na&scaron;i rezultati su pokazali da uprkos neadhezivnosti ćelija probiotskog kvasca S. boulardii, ovaj probiotik je značajno uticao na sposobnost adhezije kvasca C. glabrata. Ovaj efekat je bio veoma zavisan od sojeva vrste C. glabrata, i bio je antagonistički ili sinergistički. &Scaron;to se tiče faktora životne sredine, temperatura nije pokazala značajan uticaj na modulatorni efekat probiotskog kvasca S. boulardii, dok pri visokoj pH vrednosti, probiotski kvasac nije uspeo da potisne adheziju sojeva kvasca C. glabrata. Sličan efekat je primećen u eksperimentima sa antimikoticima, gde su povećane koncentracije antimikotika smanjile modulatorni efekat S. boulardii na adheziju C. glabrata. Sojevi vrste C. glabrata u ko-kulturi sa neadhezivnim patogenim sojevima vrsta C. krusei i S. cerevisiae su pokazali slabiju adheziju nego u ko-kulturi sa neadhezivnim probiotskim sojevima vrste S. boulardii, L. rhamnosus i L. casei. &Scaron;to ukazuje da se adhezivnost dve odvojene kulture ne može korisiti za predviđanje adhezivnosti njihove ko-kulture. Nije utvrđena korelacija između hidrofobnosti povr&scaron;ine ćelije i adhezije na polistiren povr&scaron;inu sojeva kvasca C. glabrata, &scaron;to ukazuje da hidrofobnost ćelijskog zida ne mora uvek da bude precizna mera adhezivnog potencijala.</p> / <p>Following the widespread use of immunosuppressive therapy together with broadspectrum antimycotic therapy, the frequency of mucosal and systemic infections caused by the pathogenic yeast Candida glabrata has increased in the past decades. Due to the resistance of C. glabrata to existing azole drugs, it is very important to look for new strategies helping the treatment of such fungal diseases. An increasing number of potential health benefits are being attributed to probiotic treatments. They include various bacterial probiotics, while among yeast only Saccharomyces boulardii (nom. nud.) is used extensively as a probiotic and often marketed as a dietary supplement.<br />The aim of this doctoral dissertation is to investigate the effect of the probiotic yeast S. boulardii on the adhesion of C. glabrata to polystyrene surface at different temperatures, pH values, and in the presence of three clinically important antifungal drugs, namely fluconazole, itraconazole, and amphotericin B. The cell surface hydrophobicity (CSH) of the tested C. glabrata strains has been determined as well in order to test for a possible correlation between this physico-chemical property and the ability to adhere to polystyrene surface. In addition, Candida krusei, Saccharomyces cerevisiae, two bacterial probiotics Lactobacillus rhamnosus and Lactobacillus casei were tested in a co-culture with C. glabrata as well. The method used to assess adhesion was crystal violet staining. The CSH of C. glabrata strains was determined using the Microbial Adhesion To Hydrocarbon (MATH) test. Our results showed that despite the nonadhesiveness of S. boulardii cells, this probiotic significantly affected the adherence ability of C. glabrata. This effect was highly dependent on C. glabrata strain and was either antagonistic or synergistic. Regarding the extrinsic factors, temperature did not indicate any significant influence on this S. boulardii modulatory effect, while at high pH, S. boulardii did not manage to repress the adhesion of C. glabrata strains. Similar effect was observed in the experiments with antimycotics, where increased concentrations of antimycotic reduced the modulatory effect of S. boulardii on C. glabrata adhesion. C. glabrata strains in the co-cultures with non-adhesive pathogenic strains C. krusei and S. cerevisiae showed weaker adhesion than in co-cultures with non-adhesive probiotic strains S. boulardii, L. rhamnosus and L. casei. This indicates that the adhesiveness of two separate cultures could not be used to predict the adhesiveness of their co-culture. Further, there is no correlation between cell surface hydrophobicity and adhesion of C. glabrata strains to the polystyrene surface, which indicates that the hydrophobicity of the cell wall must not always be the precise measure of the adhesive potential.</p>

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Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 100-124).

Thesis (PhD)--University of Stellenbosch, 2008. / Bibliography. Also available via the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 143-156). Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 30-37).