O fósforo (P) é limitante tanto na produtividade da cana-de-açúcar (Saccharum officinarum L.) quanto na atividade da microbiota do solo. Em solos de regiões tropicais, devido à elevada saturação por óxidos de ferro e alumínio, o P se encontra normalmente indisponível às plantas. Portanto, o manejo da adubação de P, baseado no uso de microrganismos capazes de promover uma melhor ciclagem do P, se faz necessário. Assim, o objetivo do estudo foi avaliar as mudanças na microbiota do solo e na produtividade da cana-de-açúcar, em condições de campo, em resposta ao manejo orgânico associado à inoculação de bactérias solubilizadoras de fosfato (BSF). Para tanto, foi utilizado um composto, obtido previamente por meio da compostagem de subprodutos da indústria sucroenergética (torta de filtro e cinzas), enriquecido com fosfatos de rocha (fosfato de Araxá-FA ou fosfato de Bayóvar-FB). No campo foram estabelecidos sete tratamentos, sendo um destes o tratamento controle, adubado com superfosfato triplo. Seis tratamentos compreendiam áreas adubadas com composto, sendo duas com composto que continha FA, duas com FB e duas somente com composto sem enriquecimento com P (C). A metade dessas áreas recebeu também inoculação com BSF, contendo os tratamentos FA+I, FB+I e C+I. A inoculação foi feita com Bacillus simplex BACBR04, Bacillus sp. BACBR06 e Rhizobium sp. RIZBR0. As avaliações foram realizadas durante o primeiro ano de cultivo (cana planta) em dois períodos (aos seis e 12 meses). As mudanças na microbiota foram acessadas por meio da atividade das enzimas fosfatases (ácida e alcalina), fitases e β-glucosidase. Mudanças na estrutura da comunidade bacteriana e fúngica foram acessadas por meio do T-RFLP. A abundância dos genes 16S RNAr, phoD (relacionado à solubilização de P) e ITS foram avaliadas por meio de PCR quantitativo. A atividade das fosfatases e β-glucosidase aumentaram com a inoculação na cana adubada com composto enriquecido com fosfato de Araxá (FA+I) e também no solo adubado com composto sem enriquecimento (C+I). Nos tratamentos FA+I e C+I houve incremento do fósforo disponível e aumento de 10% na produtividade, em relação ao tratamento controle (M). As comunidades bacteriana e fúngica do tratamento C+I se estruturaram de forma distinta em relação ao tratamento controle (M). Nos solos inoculados houve menor abundância do gene ITS aos seis meses, enquanto que, para o gene 16S RNAr, os solos inoculados apresentaram menor abundância no período de 12 meses. Verificou-se que o consórcio bacteriano inoculado, associado com a aplicação de composto, superou, no primeiro ano de cultivo, o manejo rotineiramente utilizado em cana-de-açúcar (adubação com superfosfato triplo). É possível que haja efeito residual no decorrer dos ciclos da cana-de-açúcar, o que ainda reforçaria a importância do manejo orgânico associado à inoculação com BSF. / Phosphorus (P) is limiting both the yield of sugarcane (Saccharum officinarum L.) and the activity of the soil microbiota. In tropical soils, due to the high saturation of iron and aluminum oxides, P is normally unavailable to plants. Therefore, the management of P fertilization, based on the use of microorganisms that promote better P cycling is necessary. Thus, the objective of this study was to evaluate changes in the soil microbiota and in sugarcane yield under field conditions, in response to the organic management associated with the inoculation of phosphate solubilizing bacteria (PSB). In our field experiment, our fertilizer was an organic compost, previously obtained by the composting process of by-products of the sugarcane industry (filter cake and ashes), enriched with rock phosphates (Araxá phosphate-AP or Bayóvar phosphate-BP). Seven treatments were established in the field, one of which was the control treatment, fertilized with triple superphosphate. Six treatments comprised compost fertilized areas, two with compost containing AP, two with BP, and two only with compost, and without any enrichment with P (C). Half of these areas were also inoculated with PSB, containing the treatments AP+I, BP+I and C+I. Field inoculation was done with Bacillus simplex BACBR04, Bacillus sp. BACBR06 and Rhizobium sp. RIZBR0. We performed evaluations during the first year of cultivation at two periods (at six and twelve months after planting). We accessed the changes in the microbiota through the activity of acid and alkaline phosphatases, phytases and β-glucosidase. Changes in bacterial and fungal community structure were accessed through T-RFLP. We evaluated the abundance of the 16S RNAr, phoD (related to P solubilization) and ITS genes by quantitative PCR. The phosphatases and β-glucosidase activity increased with the inoculation in sugarcane fertilized with compost and enriched with Araxá phosphate (AP+I) and in the soil fertilized with compost, but without any enrichment (C+I). In these treatments (AP+I and C+I), we found an increase in available phosphorus and a 10% increase in productivity, in relation to the control treatment (M). The bacterial and fungal communities of the treatments C+I presented a different structure in relation to the control treatment (M). In the soils that received bacterial inoculations, there was a lower abundance of the ITS gene at six months, whereas for the 16S RNAr gene the inoculated soils presented lower abundance at 12 months. We verified that the inoculated bacterial consortium, associated with the application of compost, overcame the conventional management in sugarcane (triple superphosphate fertilizer) in the first year of cultivation. In addition, it is possible residual effect during the sugarcane cycles, which would further reinforce the importance of organic management associated with BSF inoculation.

As antibiotic resistant bacterial strains are becoming more prevalent in healthcare settings, it is necessary to find alternative methods of detecting and treating these infections. One of the antibiotic resistant strains of interest is the carbapenem-resistant Enterobacteriaceae (CRE). CREs have the ability to evade some of the most potent antibiotics currently in use and employ carbapenemases to negate the effect of antibiotics. The three most common carbapenemase genes, found in carbapenem-resistant Enterobacteriaceae along with a gene found only in Escherichia coli were chosen to create a qPCR assay for rapid detection of resistant infections. The carbapenemase genes are KPC, VIM and NDM and the E. coli gene is uidA, a β-glucuronidase gene. Consensus sequences were obtained from each of the genes to account for the many variants of each gene. We were able to triplex the assay and test it against a library for twenty isolates varying by which gene they contain. Additional research has been conducted on the library of carbapenem-resistant Enterobacteriaceae using bacteriophages or phage. The Phage Hunters class isolated and identified twenty phage that infect K. pneumoniae. Out of the twenty phage, seven phage were able to effectively infect carbapenem-resistant K. pneumoniae.

The aim of this thesis was to determine the authenticity of fruit food for infants using molecular and instrumental methods. In the experimental part, plant DNA isolations from fruit leaves (peaches, apricots, plums and apples) and bananas were performed. Further, DNA was isolated also from five commercial products, and from model mixtures that were prepared in terms of content identical to the commercial mixtures. The isolated DNA was characterized and verified by qPCR with plant DNA-specific ITS2 primers. Three triple primer pairs were selected, and their specificity was evaluated when performing multiplex PCR. This method makes it possible to detect more types of fruit in one reaction, reducing the economic and time requirements for detection. As none of the selected primer pairs were sufficiently specific for the apricot, the evidence from the plum and peach was further realized using duplex PCR. High resolution melting curve analysis was used for better DNA type recognition. Subsequently, agarose gel electrophoresis was performed to analyse the fragment lengths. Furthermore, experiments have been made to identify some specific phenolic substances in commercial and model fruit mixtures by HPLC. Since phenolic substances are degradable under unsuitable storage conditions, the presence of individual compounds was not detected by this method.

P. aeruginosa fait partie des bactéries classées comme multirésistantes. Ce bacille est un agent pathogène opportuniste susceptible d’être présent dans les réseaux d’eau. Les contaminations sont souvent localisées au niveau des points d’usage et sont à l’origine de risques sanitaires et économiques pour les établissements de santé et les industries. Bien que différents procédés de traitements soient couramment appliqués certaines contaminations persistent sous la forme de biofilm et altèrent la qualité de l’EDCH, tout en devenant un potentiel réservoir de dissémination. L’absence de traitements efficaces et l’impact négatif des biocides sur l’environnement sont en faveur du développement de nouvelles alternatives. Les bactériophages sont exclusivement des virus de bactéries. Ces prédateurs naturels sont omniprésents dans l'environnement, ce qui nous permet de disposer d'une grande diversité et ont l’avantage des’auto-répliquer en présence de leur hôte. Dans ce contexte, cette étude évalue le potentiel de ces virus en tant qu'agents de biocontrôle pour éliminer les biofilms de P. aeruginosa. Neuf souches de P. aeruginosa, incluant la souche référence PAO1 et des souches environnementales, ont été utilisées pour étudier l'activité de neuf phages appartenant à la famille des Caudovirales. Un screening a été réalisé permettant par la méthode des spots test de sélectionner les phages les plus efficaces et les souches sensibles. Les bactéries ont ensuite été cultivées dans un milieu mineral minimum et l'efficacité des phages a été étudiée sur une culture exponentielle. Le suivi de la densité optique a permis de mettre évidence trois profils d’activités différents. Sur la base de ces résultats, deux phages et deux souches ont été conservés pour réaliser des tests sur des biofilms de 24 h implantés à la surface de coupons en INOX, représentatif des surfaces industrielles ou thermales. L’efficacité du traitement par les phages durant 14 h a été évaluée par qPCR viable. Une réduction maximale de 1,7 équivalent Log UFC.cm2 /coupon a été obtenu selon le couple étudié. Les résultats mettent également en avant une répartition des phages en faveur des cellules planctoniques, contrôlant ainsi efficacement la dissémination du biofilm dans l’environnement. Cette étude met en évidence une action des phages qui est dépendante de la souche de P. aeruginosa ainsi que de l'état physiologiques des cellules (planctoniques ou sessiles) ce qui rend difficile l’élimination d’un biofilm, même jeune. Dans le but d’améliorer l’infection de ces structures il pourrait être envisagé d'associer l'activité de plusieurs phages dans un cocktail ou de les combiner à d'autres molécules d’intérêts. / P. aeruginosa is one of the bacteria classified as multiresistant. This bacillus is an opportunistic pathogen that may be present in water networks. Contaminations are often located at the point of use and are at the origin of health and economic issues for health facilities and industries. Although different treatment processes are commonly applied, certain contaminations persist in the form of biofilm, alter the quality of EDCH and represent a reservoir of dissemination. The lack of effective treatments and the negative impact of biocides on the environment favor the development of new alternatives. Bacteriophages are exclusively bacterial viruses. These natural predators are ubiquitous in the environment, which allows us to have a great diversity, and have the advantage of self replicationin the presence of their host. In this context, this work explores the potential of these viruses as biocontrol agents to eliminate P. aeruginosa biofilms in addition to existing solutions. Nine strains of P. aeruginosa, including PAO1 reference strain and environmental strains were used to study the activity of nine phages belonging to the family Caudovirales. A screening was carried out allowing by the spottest method to select the most effective phages and sensitives trains. A screening was carried out allowing by the spot test method to select the most effective phages and sensitive strains. Bacteria were then cultivated in a mineral minimum medium and the efficiency of the phages was studied on an exponential culture phase. Monitoring of optical density has enabled to highlight three different activity profiles. On the basis of these results, two phages and two strains were kept for testing on 24-hour biofilms implanted on the surface of stainless steel coupons, representative of industrial or thermal surfaces. The efficacy of phage treatment for 14 h was evaluated by viable qPCR. A maximum reduction of 1.7 log equivalent UFC.cm2 / coupon was obtained according to the couple studied. The results also highlight a phage distribution in favor of planktonic cells, effectively controlling the release of biofilm into the environment. This study demonstrates a phage action that is dependent on the P. aeruginosa strain as well as the physiological state of the cells (planktonic or sessile). The complexity of the biofilms’ structure makes it difficult to eliminate them, even when young. In order to improve the infection of these structures it could be considered to associate the activity of several phages in a cocktail or to combine them with other molecules of interest.

Control strategies against the parasitic nematode Teladorsagia circumcincta are problematic under current sheep management systems. Infection with the parasite, particularly in young lambs, results in significant production losses therefore sustainable worm control is being sought. It has been established that variation in resistance to T. circumcincta is under genetic control and the development of resistance is an acquired characteristic and has an immunological basis. This project investigated the immunological response to infection, of lambs with predicted resistance or susceptibility to T. circumcincta. Specifically, the study aimed to identify immune response-associated genes that were differentially-expressed in resistant and susceptible lambs and attempted to identify mutations in these genes. This study was part of a long term project that aims to identify genetic marker/s to aid in marker-assisted selection (MAS) for resistance to T. circumcincta. Real time reverse transcription-quantitative polymerase chain reaction (real time RTqPCR) was performed on abomasal mucosa and lymph nodes from 55 lambs used in a previous experiment. The lambs had been either trickle-infected with 2,300 infective larvae every two days over three months (infected resistant/susceptible, n=45) or sham-dosed (non-infected control, n=10). Lambs were ranked in relation to faecal egg count (FEC) and adult worm count (AWC) at post mortem; zero or low FEC (resistant) to high FEC (susceptible). Histopathology showed only mild pathological changes in the abomasal mucosa of resistant lambs but heavy lymphocytic inflammatory infiltration in the mucosa and submucosa of infected susceptible animals. Measurements of a range of cytokine transcripts and cell markers associated with the four major CD4+ T cell subsets identified IL6, IL21, and IL23A as significantly increased by at least two-fold in abomasal lymph nodes and abomasal mucosa of susceptible lambs in comparison to resistant animals. Highly significant (P<0.02) positive correlations were found between IL6 (ρ=0.35), IL21 (ρ=0.54) and IL23A (ρ=0.38) transcript levels and AWC. Similarly, there were highly significant (P<0.01) positive correlations between FEC and IL6 (ρ=0.41), IL21 (ρ=0.65) and IL23A (ρ=0.31). In contrast, significant negative correlation (P<0.04) between IL23A with IgA antibody levels (ρ=-0.31) was found. There was also a significant positive correlation (P<0.03) of TGFB1 levels with AWC (ρ=0.42) and FEC (ρ=0.32) in the abomasal mucosa. These data suggests that susceptibility to T. circumcincta is linked to the activation of the inflammatory TH17 T cell subset and that this chronic inflammatory response was inappropriate to clear worm infection. High resolution melt analysis failed to identify single nucleotide polymorphisms in the coding regions of IL21 and IL21R. This is the first report of the involvement of TH17 response in GI worm infection in sheep. Similar gene expression studies involving the known upstream and downstream players of the TH17 response could be done.

Submitted by FERNANDA DA SILVA VON PORSTER (fdsvporster@univates.br) on 2015-08-10T17:52:45Z No. of bitstreams: 3 license_text: 21244 bytes, checksum: 51d48429f9b12e63f6b08237f8c0eabe (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) 2015HenriqueSulzbachdeOliveira.pdf: 1900803 bytes, checksum: b661d7808a3792bd808f143d5dd8e7a3 (MD5) / Approved for entry into archive by Ana Paula Lisboa Monteiro (monteiro@univates.br) on 2015-08-17T20:07:59Z (GMT) No. of bitstreams: 3 license_text: 21244 bytes, checksum: 51d48429f9b12e63f6b08237f8c0eabe (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) 2015HenriqueSulzbachdeOliveira.pdf: 1900803 bytes, checksum: b661d7808a3792bd808f143d5dd8e7a3 (MD5) / Made available in DSpace on 2015-08-17T20:07:59Z (GMT). No. of bitstreams: 3 license_text: 21244 bytes, checksum: 51d48429f9b12e63f6b08237f8c0eabe (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) 2015HenriqueSulzbachdeOliveira.pdf: 1900803 bytes, checksum: b661d7808a3792bd808f143d5dd8e7a3 (MD5) / Helicobacter pylori é uma bactéria que infecta aproximadamente 50% da população mundial, podendo causar gastrite crônica e outras formas de dano celular. A relação entre inflamação e câncer é amplamente descrita. Estímulos patogênicos induzem a expressão do fator de necrose tumoral alfa (TNF-α) que, por sua vez, induz outros mediadores responsáveis pela resposta inflamatória e proliferação celular. O presente estudo teve como objetivo avaliar a expressão dos genes TNF-α, NFKΒ1 e p38α na mucosa gástrica humana e investigar a influência da presença de H. pylori na expressão destes em uma população do Vale do Taquari, Rio Grande do Sul, Brasil. As amostras foram coletadas por endoscopia digestiva alta, sendo o diagnóstico de H. pylori realizado através do teste rápido de urease, com posterior confirmação pelo exame anatomopatológico de rotina. O RNA total foi extraído e purificado para posterior síntese de DNAc (DNA complementar) e análise por qPCR (Reação em Cadeia da Polimerase em Tempo Real). O algoritmo NormFinder foi utilizado para a análise do gene de referência. Para análise estatística dos genes de interesse foram utilizados os testes de Mann-Whitney e Kruskal-Wallis seguido pelo teste de comparações múltiplas de Dunn. Das 100 amostras coletadas, 19% foram classificadas como normal, 46% como gastrite crônica não ativa, 27% como gastrite crônica ativa e 8% como metaplasia intestinal. Todas as amostras positivas para H. pylori apresentaram inflamação ativa, de acordo com o exame anatomopatológico. Utilizou-se como gene normalizador o SDHA, que foi classificado como mais estável em relação ao ACTB, GAPDH, B2M e HPRT1. A expressão do TNF-α foi significativamente superior nos grupo H. pylori Positivo (p < 0,0001, teste de Mann-Whitney) e Gastrite Crônica Ativa (p < 0,01, Teste de Kruskal-Wallis seguido pelo teste de comparações múltiplas de Dunn). No entanto, não foi detectada diferença na expressão gênica do NFKΒ1 e do p38α entre os grupos. Pode-se concluir que a presença de H. pylori pode estar relacionada ao aumento na expressão do TNF-α, demonstrando a sua influência no processo inflamatório do tecido gástrico. Apesar de não ter sido encontrada alteração na expressão do NFKΒ1 e do p38α em nível de RNAm, estudos adicionais devem ser realizados para verificação da influência destes genes nesta via. / Helicobacter pylori infects about 50% of the world’s population, causing chronic gastritis and other forms of cellular damage. The relationship between inflammation and cancer is well known. Pathogenic stimuli induce the expression of tumor necrosis factor alpha (TNF-α), which, in turn, induce other mediators responsible for inflammation response and cell proliferation. The present study aimed to evaluate the gene expression of TNF-α, NFKB1 and p38α in human gastric mucosa and investigate the H. pylori influence in the expression of these genes in a population of Vale do Taquari, Rio Grande do Sul, Brazil. The samples were collected by upper endoscopy and the H. pylori diagnosis was performed through rapid urease test and histological analysis. The total RNA was extracted and purified for subsequent cDNA synthesis and qPCR analysis. The NormFinder algorithm was used for reference gene analysis. For the statystical analysis of the studied genes were used the Mann-Whitney test and the Kruskal-Wallis test followed by the Dunn’s test for multiple comparisons. From the 100 samples collected, 19% were classified as normal, 46% as non-active chronic gastritis, 27% as active chronic gastritis, and 8% as intestinal metaplasia. All samples positive for H. pylori demonstrated active inflammation, according to hystological analysis. SDHA was classified as the most stable gene when compared to ACTB, GAPDH, B2M and HPRT1, being chosen to be used as reference gene for qPCR normalization. The TNF-α expression was significantly higher in the H. pylori (p < 0,0001, Mann-Whitney’s test) and the Active Chronic Gastritis groups (p < 0,01, Kruskal Wallis test followed by Duncan’s multiple comparisons test). However, it wasn’t detected any difference in NFKΒ1 and p38α expression in the studied groups. It can be concluded that the presence of H. pylori can be related to TNF-α upregulation, demonstrating its influence in the inflammatory response of the gastric tissue. Although it has not been found changes in the NFKΒ1 and p38α expression at mRNA levels, additional studies are needed to verify the influence of these genes in this pathway.

Orientador: Silvio Aparecido Lopes / Resumo: Huanglongbing (HLB), causada pela bactéria Candidatus Liberibacter asiaticus (Las) que coloniza o floema das plantas cítricas, é considerada a doença mais grave e destrutiva dos citros. Aspectos da patogenicidade de Las ainda não estão compreendidos, dificultado pela condição não cultivável do patógeno. Entender como Las se movimenta internamente na planta para colonizar os diferentes tecidos é fundamental para o controle do HLB e a busca de materiais com potencial de resistência. O objetivo desse trabalho foi caracterizar a movimentação de Las em plantas de citros. Nos experimentos plantas de laranjeiras foram inoculadas por enxertia de segmentos de ramos de plantas infectadas de 2 - 3 cm de comprimento. Para estudo da velocidade de movimentação de Las nos vasos do floema, foi utilizado duas metodologias. A primeira envolveu poda sequencial do caule a diferentes distâncias do ponto de inoculação e avaliação de amostras de folhas seis meses após a inoculação. Já na segunda metodologia foi realizado a coleta de amostras de anéis de casca do caule a diferentes distâncias do ponto de inoculação e de raízes fibrosas. Foi avaliado também a influência de novos fluxos de crescimentos na movimentação de Las. Lotes de plantas foram podadas na parte aérea ou no sistema radicular para indução do crescimento. Plantas não podadas foram usadas como controle. Foram amostradas folhas que surgiram na planta após a inoculação, folhas velhas, anel de casca e raízes fibrosas a partir de 15 dias ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Huanglongbing (HLB), caused by the bacteria Candidatus Liberibacter asiaticus (Las) which colonizes the phloem of citrus plants, is considered the most serious and destructive disease of citrus. Las pathogenicity aspects are not yet understood, hampered by non-cultured condition of the pathogen. Understanding how Las moves within the plant to colonize the different tissues is fundamental for HLB management and searching for materials with resistance potential. The objective of this research was to characterize the movement of Las in citrus plants. In the experiments orange plants were inoculated using grafting segments from infected plant branches 2-3 cm long. To study the speed of Las movement in phloem, two methodologies were used. The first involved sequential pruning of the stem at different distances from the inoculation site and evaluation of leaf samples six months after inoculation. In the second methodology was collected the ring samples of stem bark at different distances from the point of inoculation until the ground level and fibrous roots. It was also evaluated the influence of the growth flows in the Las movement. Lots of plants were pruned in the aerial part or in the root system to induction of growth. Unpruned plants were used as controls. The plants were evaluated by sampling young leaves, mature leaves, stem bark and roots every 15 days from inoculation. For the study of the movement pattern of Las in the plants, we used totally, partially and non-girdling ... (Complete abstract click electronic access below) / Doutor

Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-06-02T12:47:54Z No. of bitstreams: 1 MarianaRochaMaximianoDissertacaoParcial2015.pdf: 459398 bytes, checksum: b2185a3a3375dbb7ac1ed82fc658146b (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-06-02T12:48:21Z (GMT) No. of bitstreams: 1 MarianaRochaMaximianoDissertacaoParcial2015.pdf: 459398 bytes, checksum: b2185a3a3375dbb7ac1ed82fc658146b (MD5) / Made available in DSpace on 2017-06-02T12:48:21Z (GMT). No. of bitstreams: 1 MarianaRochaMaximianoDissertacaoParcial2015.pdf: 459398 bytes, checksum: b2185a3a3375dbb7ac1ed82fc658146b (MD5) Previous issue date: 2015-06-18 / Sclerotinia sclerotiorum, the causal agent of white mold, is a plant pathogenic fungus that has a characteristic feature, production of sclerotia, resistance structures that can be viable for up to 10 years in the soil, with ability to initiate a new cycle of infection under favorable conditions. The disease control methods are based on integrated management practices, including biological and chemical control. The proteomic study of this interaction may be a strategy for studying this pathosystem, however, the small amount of mycelium produced during the infectious process in vivo greatly limits the study of this pathosystem using this strategy. The main goals of this work were the development and validation of a culture medium that would partially mimic the host and allow the production of large amounts of micelium for in vitro studies of this pathosystem. For this purpose, a protocol was established for the production of culture media prepared with leaf extract of the hosts. These media were inoculated with sclerotia of the monosporic isolate SS 200 of S. sclerotiorum and the differential expression of effectors and candidate effectors of virulence of the fungus was evaluated by qPCR. The results showed that a large amount of micelia grew in the media and effector genes and candidate effector genes were induced in these media. These results indicate that the proposed culture media can be used to study the in vitro interaction between S. sclerotiorum and several plant hosts and that they can be useful especially in proteomic studies. / Sclerotinia sclerotiorum, agente causal do mofo branco, ?? um fungo fitopatog??nico que possui uma caracter??stica marcante, a produ????o de escler??dios, estruturas de resist??ncia que podem ser vi??veis por at?? 10 anos no solo, com capacidade de iniciar um novo ciclo de infec????o em condi????es favor??veis. Os m??todos de controle da doen??a baseiam-se em pr??ticas de manejo integrado, incluindo controle biol??gico e qu??mico. O estudo prote??mico desta intera????o pode ser uma estrat??gia para o estudo deste patossistema, entretanto, a pequena quantidade de mic??lio produzido pelo pat??geno durante o processo infeccioso in vivo limita bastante o estudo deste patossistema utilizando esta estrat??gia. Os objetivos deste trabalho foram o desenvolvimento e valida????o de um meio de cultura que mimetizasse parcialmente o hospedeiro e permitisse a produ????o de grande quantidade de massa micelial para estudos in vitro desse patossistema. Para tal, foi estabelecido um protocolo para a produ????o de meios de cultura, preparados a partir de extratos foliares dos hospedeiros. Estes meios foram inoculados com escler??dios do isolado monosp??rico de S. sclerotiorum SS 200 e a express??o diferencial de genes efetores e candidatos a efetores de virul??ncia do fungo foi avaliada por qPCR. Os resultados mostraram que em todos os meios testados houve crescimento de grande quantidade de massa micelial e que os meios foram capazes de induzir a express??o de genes efetores e candidatos a efetores. Portanto, os resultados indicam que os meios propostos podem ser usados no estudo da intera????o in vitro de S. sclerotiorum com v??rios hospedeiros, especialmente em estudos prote??micos.

The extensive use of tetrachloroethene (PCE) and trichloroethene (TCE) as cleaning solvents has resulted in widespread contamination of groundwater systems with vinyl chloride (VC). VC, a known human carcinogen, is primarily formed in groundwater via incomplete anaerobic reductive dechlorination of PCE and TCE. Aerobic, methane-degrading bacteria (methanotrophs), which are capable of VC cometabolism while growing on methane, could be important in natural attenuation of VC plumes that escape anaerobic treatment. Real-time PCR (qPCR) represents an innovative approach for detecting and quantifying the presence and activity of these VC-degrading microbes. Immediate applications of this technique include use in a laboratory setting to help elucidate the potential bacterial-substrate interactions occurring in the subsurface environments at these contaminated sites; interactions that could ultimately affect the role of methanotrophs in VC degradation. This technique could also provide lines of evidence for natural attenuation of VC, thus support existing anaerobic bioremediation technologies that generate VC as a metabolic intermediate. In this work, we evaluated several PCR primer sets from the literature for use in methanotroph qPCR assays of groundwater samples. PCR primers targeting two functional genes involved in VC cometabolism, pmoA (sub-unit of particulate methane monooxygenase (pMMO)) and mmoX (sub-unit of soluble MMO (sMMO)), as well as 16S rRNA gene primers that targeted Bacteria, and Type I and Type II methanotrophs were tested. These assays were made quantitative by constructing standard curves with DNA from Methylococcus capsulatus (Type I) and Methylocystis sp. strain Rockwell (Type II). Primer sets were evaluated by comparing gene abundance estimated against known amounts of Type I and Type II methanotroph DNA. After primer validation, an effort to substantiate this methanotroph qPCR method was made by attempting to investigate methanotroph populations in groundwater samples from VC-contaminated sites. Some samples studied were also subjected to 16S rRNA gene pyrosequencing, allowing for relative abundance comparisons with qPCR analyses. Following our primer assessment experiments, effective primer sets were used to estimate the presence of methanotrophs at environmental sites in Soldotna, Alaska; Naval Air Station Oceana, Virginia Beach, Virginia; and Carver, Massachusetts. Results showed that methanotrophs were present in nearly all wells sampled from all environmental sites. Estimations of methanotroph relative abundance in environmental samples were determined by comparing the Type I and Type II primer estimates to those of the 16S universal primers. Methanotrophs in these groundwater samples ranged from 0.2% to 6.6% of the total bacterial population. Pyrosequencing analysis of the same samples showed methanotroph relative abundances that ranged from 1.7% to 54%. In groundwater samples where both DNA and RNA was extracted, the quantities of functional gene transcripts per gene copy was compared, revealing that the transcripts/gene ratio for both pmoA and mmoX was less than one, implying relatively low methanotroph activity. Analysis of mmoX environmental sample dissociation curves revealed a double peak, indicating possible non-specific PCR products. Our data suggests that most of the qPCR primer sets used in the environmental samples adequately detect methanotrophs, though the mmoX primers need to be further validated. These primer sets will be useful for supporting VC bioremediation strategies by providing a rapid, convincing, and cost effective alternative the enrichment culture technique currently employed. Comparison of qPCR and pyrosequencing analysis revealed biases in either one, or both techniques. Finally, our preliminary transcripts/gene data suggests that the methanotrophs at the Carver site are not actively expressing pMMO and sMMO genes above basal levels.

En la producción de vinagres no se cuenta con cultivos iniciadores en los que se conozca su composición microbiológica y tampoco se conocen las propiedades oxidativas que presentan. La falta de cultivos iniciadores en la producción de vinagres se ve reflejada en una iniciación lenta de la acetificación o en paradas de acetificación. La utilización de inóculos permite, a la vez, la uniformización en la calidad final del producto, asegurando los parámetros de calidad predeterminados. Las bacterias acéticas son los microorganismos responsables de la biotransformación de vino a vinagre gracias a su capacidad para oxidar el etanol a ácido acético y acumularlo en el medio sin gran toxicidad para ellas. Esta oxidación tiene lugar en dos pasos iniciándose por la acción de la enzima ADH (Alcohol deshidrogenasa). Este trabajo estudia las diferencias en la expresión de la ADH-PQQ en medios con diferentes fuentes de carbono. Las diferencias en la expresión pueden ser utilizadas como indicador de la capacidad acetificadora de diferentes especies de bacterias acéticas. Se propone el uso de la RT-QPCR (real-time quantitative reverse transcription polymerase chain reaction), como técnica para cuantificar la expresión relativa de la ADH. Esto permite poner a punto la metodologia para el análisis de la expresión relativa de la ADH y aplicarla posteriormente a otros genes de las bacterias acéticas. Se logró establecer la relación entre la actividad de la ADH con la producción de ácido acético en la producción de vinagres. Esta relación se observo especialmente en el género Acetobacter. Siendo la primera vez que se utiliza esta técnica en el área de las acetificaciones, proponemos esta técnica como una herramienta para el estudio del metabolismo de las bacterias acéticas.