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Made available in DSpace on 2014-08-20T13:32:46Z (GMT). No. of bitstreams: 1 tese_marcia_magalhaes_mata.pdf: 1998479 bytes, checksum: c18127c54931180d99c33eb9ceb106cd (MD5) Previous issue date: 2013-06-24 / L. monocytogenes is the etiologic agent of listeriosis, a severe food-borne disease. This pathogen has also variable ability to adhere to food-processing surfaces. Thus, the aims of this study was at first to evaluate the influence of the temperature (04-10-25-37°C) and time of incubation (24-48-168h) on the formation of attached cells by L. monocytogenes strains of diverse origins, serotypes and lineages using a colorimetric microtitre plate method. After this, comprehensive proteomics experiments using label-free 1D- liquid chromatography/tandem mass spectrometry (1D-LC/tandem MS) were performed to determine if the global proteomic responses of L. monocytogenes strains (Siliken and F2365) is altered markedly as attached cells compared to its planktonic state when growth media and temperature are the same. Our results showed that attached cells produced by different origins of L. monocytogenes did not change significantly when subjected to experimental conditions, unlike what was observed with attached cells produced by different serotypes and lineages of L. monocytogenes, which were clearly affected by environmental conditions.such as temperature and time of incubation. The ability of lineage II and serotype 1/2a and 1/2b to form large amount of attached cells when compared with the others in specific conditions indicates that risks from Listeria adherence must be taken seriously in sensitive food environments in order to find safer alternatives to prevent contamination and further dissemination of listeriosis. Only 8 proteins demonstrated substantial changes in common between both strains and temperatures in attached cells compared to their planktonic counterparts. They are: GroEL, DnaK, PtsH, PdxS, Pgi, RpsB, RpsD, and RpsP. Moreover, it was observed that the cell surface protein BapL abundance, though low, was not enhanced in attached cells suggesting its role in adherence could be a generalized contribution to the cell wall hydrophobicity. Interestingly, our experiment suggest that at 25°C the attached cells in both strains undergo flagella synthesis repression. Also, Sig B Regulon can be associate with an enhanced general stress response occurs in lineage II Strain (Siliken) but not in lineage I Strain (F2365) and could relate to the consequences of attachment. The temporal survey-based approach demonstrates clearly that high coverage represents a powerful means to investigate dynamic responses in L. monocytogenes from a functional genomics perspective / L. monocytogenes é o agente etiológico da listeriose, uma doença severa de origem alimentar. Esse patógeno também é capaz de se aderir a uma grande variedade de superfícies do processamento de alimentos. Sendo assim, os objetivos deste estudo foram primeiramente, avaliar a influência da temperatura (04-10-25 e 37°C) e tempo de incubação (24-48-168h) na formação de células aderidas de cepas de L. monocytogenes de diferentes origens, sorotipos e linhagens utilizando o método colorimétrico em placas de microtitulação. Após, experimentos de proteômica abrangentes que não utilizam marcadores, como a Cromatografia líquida de 1D/ espectrometria de massa em tandem (1D-LC/tandem MS) foram realizadas para determinar se o perfil proteico global de células planctônicas e de células aderidas de cepas de L. monocytogenes (Siliken e F2365) foi alterado significativamente quando os meios de crescimento e de temperatura de incubação foram os mesmos. A partir dos resultados obtidos verificou-se que as células aderidas formadas por L. monocytogenes de diferentes origens não sofreram alterações significativas quando submetidas às condições experimentais, diferentemente do que foi observado com as células aderidas formadas por L. monocytogenes de diferentes sorotipos e linhagens, as quais foram claramente afetadas pelas condições do ambiente. A habilidade da linhagem II e dos sorotipos 1/2a e 1/2b de formar grande quantidade de células aderidas quando comparadas com as demais, em condições específicas, indica alto risco de contaminação e disseminação da listeriose, bem como a sobrevivência e persistência deste micro-organismo no ambiente. Com base na análise proteômica, apenas oito proteínas demonstraram alterações substanciais em comum entre ambas as cepas e temperaturas em células aderidas comparadas com suas respectivas células planctônicas. São elas: GroEL, DnaK, PtsH, PdxS, Pgi, RpsB, RpsD, and RpsP. Verificou-se também que a abundância da proteína de superfície celular BapL, embora baixa, não foi aumentada em células aderidas sugerindo que o seu papel na adesão pode ser uma contribuição generalizada para a hidrofobicidade da parede celular. De forma muito interessante, nosso experimento sugere que células aderidas a 25°C por ambas as cepas levam a uma síntese de repressão flagelar. E ainda, Sig B Regulon pode estar associado com o aumento em geral da resposta ao estresse ocorrido na cepa de linhagem II (Siliken) mas não na cepa de linhagem I (F2365) o que pode estar relacionado com as consequências da adesão. A técnica utilizada no experimento demonstrou claramente que com alta abrangência é possível estudar proteomas bacterianos representando assim uma ferramenta poderosa para investigar respostas dinâmicas em L. monocytogenes através de uma perspectiva de genômica funcional.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-04-10T13:40:20Z No. of bitstreams: 1 fabianotouzdjianpinheirotavora.pdf: 2717332 bytes, checksum: ef7258702d1bc7c43c78dae666bfb284 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-04-11T11:35:09Z (GMT) No. of bitstreams: 1 fabianotouzdjianpinheirotavora.pdf: 2717332 bytes, checksum: ef7258702d1bc7c43c78dae666bfb284 (MD5) / Made available in DSpace on 2017-04-11T11:35:09Z (GMT). No. of bitstreams: 1 fabianotouzdjianpinheirotavora.pdf: 2717332 bytes, checksum: ef7258702d1bc7c43c78dae666bfb284 (MD5) Previous issue date: 2017-03-03 / A bactéria fitopatogênica Xanthomonas campestris pv. campestris (Xcc) consiste no agente causal da podridão negra que acomete todas as variedades comerciais do gênero Brassica, sendo responsável por perdas significativas na brassicultura nacional e mundial. O objetivo deste trabalho consistiu em traçar o perfil proteômico de dois isolados de Xcc, distintos quanto à virulência, utilizando um sistema in vitro para a identificação de proteínas diferencialmente abundantes relacionadas aos mecanismos de virulência. Inicialmente, curvas de crescimento bacteriano foram desenvolvidas afim de se determinar o momento apropriado para a coleta e extração de proteínas. Os isolados Xcc51 e XccY21 foram cultivados em dois meios de cultura distintos, sendo um nutricionalmente rico (NYG) e outro capaz de induzir a transcrição de genes relacionados à virulência (meio mínimo – XVM1), até atingirem a fase exponencial máxima de crescimento bacteriano (OD600nm = 0,8 em meio NYG e OD600nm = 0,58 em meio XVM1). As proteínas totais dos isolados foram extraídas usando fenol, precipitadas com acetato de amônio em metanol, digeridas com tripsina e analisadas pela técnica 2DnanoUPLC/MSE, utilizando a plataforma de identificação e quantificação de proteínas Protein Lynx Global Server (PLGS). Os dados obtidos foram comparados com sequências depositadas no banco de dados XGB (The Xanthomonas Genome Browser). Foram identificadas mais de 600 proteínas no proteoma total de ambos os isolados de Xcc, revelando a expressão de proteínas exclusivas, bem como um perfil diverso de proteínas aumentadas e diminuídas entre os isolados durante o cultivo nos dois meios. Com o auxílio do software BLAST2GO, foi realizada uma análise baseada na ontologia dos produtos gênicos. Os resultados obtidos evidenciam que durante a condição de indução de genes de patogenicidade, o isolado mais virulento Xcc51 se destacou pela abundância de importantes proteínas relacionadas à infecção bacteriana, como bfeA, TonB, ompP6, clpB, tig, acvB, quando comparado à quantidade exibida no isolado XccY21. Os dados proteômicos gerados pelo presente estudo apresentaram um interessante rol de proteínas diferencialmente aumentadas e supostamente relacionadas à patogenicidade e virulência de Xcc, o qual poderá nortear futuras investigações quanto aos mecanismos moleculares envolvidos na patogênese dessa fitobactéria. / The phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc) consist in the black rot disease causal agent, which affects all Brassica genus commercial varieties. This disease results in significant losses for brassicultures worldwide. The present study aimed to analyze the proteomic profile of two distinct Xcc isolates, employing an in vitro system for the identification of differentially abundant proteins, related to pathogenicity and virulence mechanisms. Initially, bacterial growth curves were assessed to determine the appropriate stage for protein extraction. Xanthomonas isolates Xcc51 and XccY21 were cultured in two distinct mediums, a nutritionally rich medium (NYG) and a minimal medium - XVM1, capable of inducing the transcription of pathogenicity genes, until they reached maximum exponential growth phase (OD600nm = 0.8 A. and OD600nm = 0.52 A., respectively). Total proteins were extracted using phenol/ammonium acetate, trypsin digested and analyzed by 2DnanoUPLC/MSE, coupled with Protein Lynx Global Server (PLGS). Xanthomonas Genome Browser database was used, leading to the identification of more than 600 proteins and revealing the expression of unique proteins as well as diverse profiles of increased and decreased proteins. Blast2GO software was applied to analyze gene ontology. Results suggest that during pathogenicity inducing condition, the isolate Xcc51 increases the abundance of crucial infection-related proteins such as bfeA, TonB, ompP6, clpB, acvB, when compared to XccY21 which could explain its higher virulence. The proteomic data showed by the present study delivered a list of interesting proteins presumably related with pathogenicity and virulence of Xcc, which could guide future investigations on molecular mechanisms involved in this phytobacterium pathogenesis.

Le tractus digestif et le foie interagissent continuellement, non seulement à travers les connexions anatomiques, mais également par des liens physiologiques/fonctionnels. Le déséquilibre de l’axe intestin-foie apparait de plus en plus comme un facteur primordial dans les désordres cardiométaboliques, à savoir l’obésité, le syndrome métabolique, le diabète de type 2 et la stéatose hépatique non alcoolique (NAFLD), pour lesquels la prévalence demeure alarmante, les mécanismes moléculaires encore méconnus, et les traitements peu efficaces. L’hypothèse centrale du présent projet de recherche est que la combinaison d’anomalies génétiques et nutritionnelles affecte la sensibilité de l’insuline intestinale, ce qui conduit à une surproduction des chylomicrons, à une dyslipidémie, une insulinorésistance systémique et des répercussions sur le foie. Dans cet agencement, le foie développe une NAFLD progressive, impliquant plusieurs sentiers métaboliques intrinsèques et des mécanismes comprenant le stress oxydatif, l’inflammation et l’insulinorésistance. En revanche, des nutriments, comme les acides gras polyinsaturés (AGPI) n-3, peuvent présenter des effets bénéfiques en ciblant plusieurs circuits pathogéniques. L’objectif central de cette thèse consiste à : (i) Démontrer que des gènes codant pour les protéines intestinales clés associées au transport des lipides, comme c’est le cas du Sar1b GTPase, peuvent interagir avec l’environnement nutritionnel pour produire l’obésité et des dérangements cardiométaboliques, incluant la NAFLD ; (ii) Explorer les mécanismes hépatiques sous-jacents à la NAFLD; et (iii) Identifier les effets et les cibles thérapeutiques des AGPI n-3 sur la NAFLD. Ces objectifs seront soutenus par une prospection de la littérature scientifique disponible dans les champs du syndrome métabolique et de la NAFLD afin d’en disséquer les forces et les faiblesses au bénéfice de la communauté scientifique. À ces fins, nous avons utilisé des modèles animaux et cellulaires manipulés génétiquement, des animaux exposés de façon chronique à des diètes riches en lipides, des spécimens de tissus hépatiques obtenus durant la chirurgie bariatrique d’obèses morbides, et une cohorte d’adolescents obèses souffrant de NAFLD et qui seront traités avec les AGPI n-3. L’ensemble de nos expériences ont soutenu nos hypothèses et ont mis en évidence les concepts et mécanismes suivants : (i) L’abondance d’un gène crucial (notamment Sar1b GTPase) au niveau de l’intestin, en synergie avec une alimentation obésogène, perturbe l’homéostasie locale et mène à des dérangements cardiométaboliques, défiant même l’axe intestin-foie ; (ii) Les causes développementales de la NAFLD comprennent les dérangements du métabolisme des acides gras, du statut redox et inflammatoire, de la sensibilité à l’insuline, des sentiers métaboliques (lipogenèse, β-oxydation, gluconéogenèse) et de l’expression des facteurs de transcription; et (iii) Les AGPI n-3 représentent un robuste arsenal thérapeutique des dérangements cardiométaboliques, notamment la NAFLD, en agissant sur plusieurs cibles pathogéniques. Globalement, nos résultats montrent le rôle indéniable de l’intestin comme organe insulino-sensible interagissant de près avec les aliments et capable de déclencher des troubles métaboliques. Plusieurs mécanismes gouvernant les désordres métaboliques ont été dévoilés par nos travaux. En outre, nos études cliniques ont pointé la force thérapeutique des AGPI n-3 qui interviennent dans de nombreux processus de régulation métaboliques et notamment dans le stress oxydatif et l’inflammation. / The digestive tract and liver interact continuously, not only through anatomical connections, but also through physiological / functional links. The imbalance of the intestine-liver axis is increasingly emerging as a key factor in cardiometabolic disorders (CMD), namely obesity, metabolic syndrome, type 2 diabetes, and non alcoholic fatty liver disease (NAFLD), for which prevalence remains alarmingly high, molecular mechanisms are poorly understood, and treatments are largely inefficient. The central hypothesis of this research project is that the combination of genetic and nutritional abnormalities affect intestinal insulin sensitivity, leading to overproduction of chylomicrons, dyslipidemia, systemic insulin resistance and dysregulated intestine-liver axis. In this situation, the liver develops progressive NAFLD, implicating several intrinsic metabolic pathways and mechanisms, including oxidative stress, inflammation and insulin resistance. In contrast, functional foods, such as omega-3 polyunsaturated fatty acids (n-3 PUFA), may have beneficial effects by targeting several pathogenic pathways. The central objective of this thesis is to: (i) Demonstrate that genes coding for key intestinal proteins associated with lipid transport, as is the case with Sar1b GTPase, can interact with the nutritional environment to produce obesity and CMD, including hepatic steatosis; (ii) explore the mechanisms underlying NAFLD; and (iii) identify the effects and therapeutic targets of n-3 PUFA. These objectives will be supported by a critical review on metabolic syndrome and NAFLD in order to dissect their strengths and weaknesses for the benefit of the scientific community. For these purposes, we used genetically engineered animal and cell models, chronic exposure of animals to high-fat diets, liver tissue specimens obtained during bariatric surgery of morbidly obese patients, and treatment of obese NAFLD adolescents with n-3 PUFA. All of our experiments supported our hypotheses and highlighted the following concepts and mechanisms: (i) The abundance of a crucial gene (notably Sar1b GTPase) in the intestine, in synergy with an obesogenic diet, disrupts local homeostasis and leads to CMD, challenging even the intestine-liver axis; (ii) Developmental causes of NAFLD include disturbances of fatty acid metabolism, redox and inflammatory status, insulin sensitivity, metabolic pathways (lipogenesis, β-oxidation, gluconeogenesis), and expression of transcription factors; and (iii) n-3 PUFA represent a robust therapeutic arsenal of CMD, including NAFLD, by acting on several pathogenic targets. Overall, our results show the undeniable role of the intestine, as an insulin-sensitive organ, interacting closely with obesogenic food, and capable of triggering CMD, including perturbations of the intestine-liver axis. Several mechanisms governing metabolic disorders have been unveiled by our work. In addition, our clinical studies have pointed to the therapeutic potential of n-3 PUFA involved in many regulatory processes, including oxidative stress and inflammation.