Mobile group II introns are retroelements that insert site-specifically into DNA target sites by a process called retrohoming. Retrohoming is mediated by a ribonucleoprotein particle (RNP) that contains both the intron RNA and the intronencoded protein (IEP). My dissertation focuses on two mobile group II introns: Lactococcus lactis Ll.LtrB and Escherichia coli EcI5, which belong to structural subclasses IIA and CL/IIB1, respectively. Previous studies showed that the Ll.LtrB IEP, denoted LtrA protein, is pole localized in E. coli. First, I found that active LtrA protein is associated with E. coli membrane fractions, suggesting that LtrA pole localization might reflect association with a membrane receptor. Second, I found that EcI5 is highly active in retrohoming in E. coli and obtained a comprehensive view of its DNA target site recognition by selection experiments. I found that EcI5 recognizes DNA target sequences by using both the IEP and base pairing of the intron RNA, with the IEP having different target specificity than for other mobile group II introns. A computer algorithm based on the empirically determined DNA recognition rules enabled retargeting of EcI5 to integrate at ten different sites in the chromosomal lacZ gene at frequencies up to 98% without selection. Finally, I developed methods for gene targeting in the frog Xenopus laevis by using Ll.LtrB RNPs for site-specific DNA modification in isolated sperm nuclei, followed by in vitro fertilization to generate genetically modified animals. The site-specific integrations were efficient enough to detect in fifty sperm nuclei for a multiple copy target site, the Tx1 transposon, and several hundred sperm nuclei for protein-encoding genes. Based on these results, I obtained transgenic tadpoles with sitespecific Tx1 integrations by simple screening. To facilitate screening for embryos with targeted integrations in protein-encoding genes, I constructed an intron carrying a GFPRAM (Retrotransposition-Activated Marker). By using this GFP-RAM with introns containing randomized sequences that base pair with the target DNA, I obtained tadpoles with intron integrations at different genomic locations, including protein-encoding genes. The methods for using group II introns for targeted sperm DNA modification in X. laevis may be applicable to other animals. / text

Submitted by Suzana Kotzent (su_kotzent@hotmail.com) on 2017-03-17T13:27:02Z No. of bitstreams: 1 Dissertação final 170317 Suzana Kotzent.pdf: 1627152 bytes, checksum: fb879e427474125c781d95d8fcc0faa5 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2017-03-23T16:43:06Z (GMT) No. of bitstreams: 1 kotzent_s_me_jabo_par.pdf: 1254030 bytes, checksum: 86a63f13335a097462b3bb9d50cce954 (MD5) / Made available in DSpace on 2017-03-23T16:43:06Z (GMT). No. of bitstreams: 1 kotzent_s_me_jabo_par.pdf: 1254030 bytes, checksum: 86a63f13335a097462b3bb9d50cce954 (MD5) Previous issue date: 2017-02-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os probióticos são microorganismos vivos que afetam de forma benéfica o hospedeiro ou o ambiente. Na aquicultura podem ser usados tanto na água como na ração, mas seu uso na alimentação é destacado como uma das principais medidas profiláticas. As doenças bacterianas são consideradas um dos principais entraves no crescimento da aquicultura, e assim, há a necessidade urgente no desenvolvimento de probióticos. O tambaqui Colossoma macropomum é a espécie nativa mais produzida no Brasil, e apesar de sua importância econômica, não há estudos que estabeleçam os microorganismos com potencial probiótico para esta espécie de peixe. Neste trabalho foi possível identificar e caracterizar bactérias autóctones com potencial probiótico para o tambaqui a partir de testes de: caracterização morfológica, catalase, tolerância à bile, antagonismo frente à patógenos, sensibilidade a antimicrobianos e sequenciamento do gene 16S rRNA. As cepas selecionadas foram: Enterococcus faecalis, Enterococcus hirae, Lactococcus lactis, Pediococcus pentosaceus, Staphylococcus hominis e Staphylococcus saprophyticus. Todas as cepas foram tolerantes aos ácidos biliares do tambaqui e capazes de inibir o crescimento dos patógenos Enterococcus casseliflavus, Lactococcus garvieae e Aeromonas hydrophila. Todas as cepas foram parcialmente resistentes contra sete antibióticos. Como as cepas de S. saprophyticus e E. faecalis apresentaram menores valores no teste de antagonismo e por estas bactérias serem relatadas como agentes zoonóticos, concluímos este estudo selecionando quatro potenciais cepas: E. hirae, L. lactis, P. pentosaceus, S. hominis. Este é o primeiro estudo a referir o potencial uso probiótico de cepas autóctones para o tambaqui. / Probiotics are living microorganisms that beneficially affect the host or the environment. In aquaculture it can be used in both water and feed, but its use in feed is highlighted as one of the main prophylactic measures. Bacterial diseases are considered to be one of the major obstacles to the growth of aquaculture, and thus, there is an urgent need for the development of probiotics. The tambaqui Colossoma macropomum is the most produced native species in Brazil, and despite its economic importance, there are no studies that establish the microorganisms with probiotic potential for this fish species. In this study it was possible to identify and characterize autochthones bacteria with probiotic potential for tambaqui from tests of: morphological characterization, catalase, bile tolerance, antagonism of pathogens, antimicrobial susceptibility and 16S rRNA gene sequencing. The selected strains were: Enterococcus faecalis, Enterococcus hirae, Lactococcus lactis, Pediococcus pentosaceus, Staphylococcus hominis and Staphylococcus saprophyticus. All strains were tolerant to acids bile from tambaqui and capable of inhibiting the growth of Enterococcus casseliflavus, Lactococcus garvieae and Aeromonas hydrophila pathogens. All strains were partially resistant against seven antibiotics. Since the strains of S. saprophyticus and E. faecalis presented lower values in the test of antagonism and because these bacteria were reported as zoonotic agents, we conclude this study selecting four potential strains: E. hirae, L. lactis, P. pentosaceus, S. hominis. This is the first study to mention the potential probiotic use of autochthonous strains for tambaqui.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1949. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 136-145).

Techn. University, Diss., 2005--München.

Thèse (Ph.D)--Université Laval, 2003. / Titre de l'écran-titre (visionné le 22 mars 2004). Bibliogr.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-11-06T10:14:45Z No. of bitstreams: 1 texto completo.pdf: 1239459 bytes, checksum: e1a7cd2486c370dcab988719055849df (MD5) / Made available in DSpace on 2017-11-06T10:14:45Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1239459 bytes, checksum: e1a7cd2486c370dcab988719055849df (MD5) Previous issue date: 2017-06-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A utilização de cepas fermentadoras de Lactococcus lactis subsp. lactis produtoras de nisina em alimentos fermentados tem vantagem para a indústria pois permite um controle adicional de contaminantes, como o patógeno de origem alimentar Listeria monocytogenes. No entanto, as interações microbianas devem ser avaliadas para garantir a produção da bacteriocina no alimento e o efeito na população do patógeno. L. monocytogenes tem a capacidade de resistir a diversas condições de estresse encontradas no alimento e durante o processamento, expressando diferentes genes como o fator siga alternativo (sigB), a enzima glutamato descarboxilase (gadD), a chaperona GroEL e o transportador de glicina betaína (gbu). A exposição a uma condição de estresse em nível subletal é capaz de conferir uma maior resistência a L. monocytogenes de sobreviver a outras situações. Nesse contexto, o objetivo deste trabalho foi avaliar a expressão de genes de estresse de L. monocytogenes em interação com L. lactis subsp. lactis produtor de nisina em meio de cultura e leite. A produção de nisina em caldo BHI e leite foi avaliada por sua detecção no sobrenadante do meio de crescimento e pela expressão do gene nisK. A expressão dos genes de estresse de L. monocytogenes sigB, gadD2, groEL e gbu foi avaliada relativamente a cultura pura e na interação com L. lactis subsp. lactis. A expressão relativa dos genes de estresse de L. monocytogenes foi variável. No entanto, a expressão dos genes sigB, groEL e gbu foi inferior no tempo de 24 h durante a interação, em relação a cultura pura, o que pode indicar uma menor capacidade de sobrevivência aos estresses quando a bactéria se encontra em interação com L. lactis subsp. lactis produtor de nisina. / The use of nisin producing fermentative strains of Lactococcus lactis subsp. lactis in fermented foods has an advantage for the industry because it allows an additional control of contaminants, such as the food-borne pathogen Listeria monocytogenes. However, microbial interactions should be evaluated to ensure bacteriocin production in the food and the effect on the pathogen population. L. monocytogenes has the ability to withstand the various stress conditions encountered in food and during processing, expressing different genes such as the alternative sigma factor (sigB), the glutamate decarboxylase enzyme (gadD), the GroEL chaperone and the glycine betaine transporter (gbu). The exposure to a stress condition at the sublethal level is able to confer a greater resistance to L. monocytogenes to survive other situations. In this context, the aim of this work was to evaluate the expression of L. monocytogenes stress genes in interaction with nisin producing L. lactis subsp. lactis in culture medium and milk. The production of nisin in BHI broth and milk was evaluated by its detection in the supernatant of the growth medium and by the expression of the nisK gene. Expression of sigB, gadD2, groEL and gbu stress genes of L. monocytogenes was evaluated for pure culture and for the interaction with L. lactis subsp. lactis. The relative expression of the L. monocytogenes stress genes was variable. However, expression of the sigB, groEL and gbu genes was lower at 24 h during interaction than in pure culture, which may indicate a lower ability to survive stress when the bacterium is interacting with nisin producing L. lactis subsp. lactis.

Cheese is a fermented product in which bacteria contribute to different flavours and textures. In order to understand the microbial processes in cheese, it is necessary to not only look at the genomic information in bacteria. The metabolome consists of a complete collection of metabolites in a biological sample. These metabolites are small molecules with a Mr >1.5 kDa, including flavour compounds. During the ripening process of cheese, many microbiological and biochemical changes occur that give cheese a diversity of textures and flavours. Proteins that go through proteolysis and amino acid catabolism are of great importance in the development of flavour in cheese, regardless of variety. Even though techniques for measurements of metabolites have existed for a long time, there are some unique challenges by analysing of several metabolites in parallel in a biological sample that promotes different metabolic pathways. Metabolic fingerprinting is the most common approach used in metabolomics, which is based on statistical analysis that through algorithms presents differences between samples. The electronic nose is able to identify the sum of volatile metabolites in a food, which is unlike the gas chromatograph that identifies individual metabolites. The aim of this review is to evaluate the use of metabolomics of selected Enterobacteriaceae together with electronic nose technology in order to analyse possible patterns of volatile metabolites produced in soft cheese. By this we hope to evaluate potential application of this approach in food quality control and microbial contamination screening. The pilot study was done together with the center for AASS, Örebro University where bacteria were analysed using the electronic nose NST3320. The study showed that it is possible to discriminate between Enterobacteriaceae, Staphylococcus aureus and cheese-associated bacteria, but also between the Enterobacteriaceae species Escherichia coli, Hafnia alvei and Klebsiella neumoniae. It is important to consider the gas sensors gradually lose their ability to detect substances after continual use, in which they need to be replaced with new gas sensors. Further, data processing requires special knowledge and can be hard to handle if the expertise is lacking. We believe that there is evidence that metabolomics together with the electronic nose have future prospects in terms of quality control and microbial contamination screening.

Cheese is a fermented product in which bacteria contribute to different flavours and textures. In order to understand the microbial processes in cheese, it is necessary to not only look at the genomic information in bacteria. The metabolome consists of a complete collection of metabolites in a biological sample. These metabolites are small molecules with a Mr >1.5 kDa, including flavour compounds. During the ripening process of cheese, many microbiological and biochemical changes occur that give cheese a diversity of textures and flavours. Proteins that go through proteolysis and amino acid catabolism are of great importance in the development of flavour in cheese, regardless of variety. Even though techniques for measurements of metabolites have existed for a long time, there are some unique challenges by analysing of several metabolites in parallel in a biological sample that promotes different metabolic pathways. Metabolic fingerprinting is the most common approach used in metabolomics, which is based on statistical analysis that through algorithms presents differences between samples. The electronic nose is able to identify the sum of volatile metabolites in a food, which is unlike the gas chromatograph that identifies individual metabolites. The aim of this review is to evaluate the use of metabolomics of selected Enterobacteriaceae together with electronic nose technology in order to analyse possible patterns of volatile metabolites produced in soft cheese. By this we hope to evaluate potential application of this approach in food quality control and microbial contamination screening. The pilot study was done together with the center for AASS, Örebro University where bacteria were analysed using the electronic nose NST3320. The study showed that it is possible to discriminate between Enterobacteriaceae, Staphylococcus aureus and cheese-associated bacteria, but also between the Enterobacteriaceae species Escherichia coli, Hafnia alvei and Klebsiella pneumoniae. It is important to consider the gas sensors gradually lose their ability to detect substances after continual use, in which they need to be replaced with new gas sensors. Further, data processing requires special knowledge and can be hard to handle if the expertise is lacking. We believe that there is evidence that metabolomics together with the electronic nose have future prospects in terms of quality control and microbial contamination screening.

Les souches de Lactococcus lactis ssp. cremoris sont caractérisées par un polymorphisme génétique conduisant à des propriétés technologiques différentes. Le but de ce projet était d'évaluer la diversité génétique des souches de L. lactis ssp. cremoris par deux approches moléculaires. D'une part, l'hybridation génomique comparative des souches combinée à l'analyse MLSA ont permis de classifier les souches et d'identifier des marqueurs pour la sélection et la distinction des souches de ferments. D'autre part, l'analyse comparative des transcriptomes des souches au cours d'une simulation des conditions de fabrication du Cheddar a permis d'identifier deux types de réponses, a) un transcriptome commun qui pourrait représenter une réponse commune de toutes les souches de Lactococcus lactis ssp. cremoris au cours de la fabrication, et b) des réponses spécifiques pour chacune des souches pouvant les distinguer. Celles-ci permettront d'identifier des marqueurs technologiques qui seront utilisés en industrie, tels que les transporteurs d'oligopeptides, des gènes de la résistance, et d'autres gènes liés à la production de flaveurs. L'analyse de la diversité génétique entre les souches de L. lactis ssp. cremoris a permis de mieux comprendre les origines de la diversité et d'identifier des gènes potentiellement liés à la différence des comportements et de la réponse des souches

Les ferments utilisés dans la fabrication du fromage Cheddar, généralement composés de combinaisons de souches de Lactococcus lactis ssp. cremoris, ont un impact important sur la qualité du produit final. Une dynamique d’association est susceptible de s’installer entre les souches présentes. L’objectif de cette étude visait à distinguer les différents groupes de souches de L. lactis ssp. cremoris sélectionnées et à quantifier les proportions de chaque groupe de souches utilisées comme ferment afin d’étudier leur comportement lors d’une fabrication de fromage Cheddar. Lors de l’analyse MLST, le gène nusA s’est démarqué des autres car il a permis de séparer les 23 souches en six groupes intéressants. Une méthode PMA-qPCR spécifique a donc été développée avec ce gène pour cible. Cette technique a été utilisée pour l’évaluation des proportions des souches en combinaisons pendant une simulation de production fromagère. Cette méthode pourrait avantageusement être transférée à l’industrie puisqu’elle est accessible, rapide et reproductible. / Starters used in Cheddar cheesemaking have a major impact on the quality of the final product. These starters are usually composed of Lactococcus lactis subsp. cremoris strain combinations and interactions between strains can be present. The main goal of this study was to discern groups of L. lactis subsp. cremoris and to quantify proportions of each strain group with the objective of studying their behavior during Cheddar cheesemaking. The MLST study showed that the nusA gene classifies strains into six interesting groups. Therefore, a specific PMA-qPCR method was developped with this target gene. The technique was used to quantify strain proportions during cheesemaking simulation. This method could be transferred to the industry because it is easy to use, fast and reproductible.