O mercúrio (Hg) é um metal não essencial, encontrado em diferentes formas químicas, com propriedades bastante diferentes. Entretanto, todas são tóxicas e capazes de alterar inúmeras vias metabólicas. Dentre os efeitos tóxicos da exposição ao Hg, tem grande relevância a toxicidade sobre os tecidos renal, cerebral e hepático. Os principais efeitos nefrotóxicos decorrentes da exposição ao Hg têm sido atribuídos ao estresse oxidativo e produção de espécies reativas do oxigênio (ROS), além de causar danos no metabolismo mitocondrial, alterando a funcionalidade da membrana celular e a polaridade tubular renal. As formas orgânicas do Hg apresentam efeitos tóxicos sobre o sistema nervoso central (SNC), com a capacidade de induzir sérios danos ao desenvolvimento neurológico, tais como deficiências na fala, hiperatividade, dificuldade de concentração e até mesmo desordens semelhantes ao autismo. No tecido hepático, os principais efeitos tóxicos ocorrem por alterações no ciclo entero-hepático, processo-chave na excreção do mercúrio. A exposição aos metais, de uma maneira geral, ativa um mecanismo que modifica a produção alterada de proteínas na tentativa de manter a homeostase e de proteger ou reparar macromoléculas que são alvos diretos ou indiretos de xenobióticos. Neste sentido, o objetivo deste trabalho foi avaliar, por meio de mapas bidimensionais, o perfil de expressão de proteínas em amostras fígado, cérebro e rim de ratos expostos a diferentes espécies de mercúrio (inorgânico, metilmercúrio e etilmercúrio) comparados aos seus respectivos controles, utilizando a plataforma clássica de análise proteômica (2DE e MS). Observou-se que a exposição às diferentes formas de Hg modificou a expressão de 139 proteínas, sendo 22 em células cerebrais, 19 em células do tecido renal, e 27 em células do tecido hepático. Dentre as proteínas diferencialmente expressas, destacaram-se àquelas relacionadas diretamente ao estresse oxidativo, nos tecidos hepático e renal e relacionadas à homeostase do Ca+2 e à regulação de glutamato na fenda sináptica, no tecido cerebral. Este estudo demonstrou que todas as formas de Hg, promoveram mudanças no perfil protéico em todos os tecidos e, muitas proteínas alteradas, corroboram com os mecanismos de ação do Hg, já previstos na literatura / Mercury (Hg) is not essential metal found in various chemical species with different properties, but all are toxic and can alter numerous metabolic pathways. Among the toxic effects of exposure to mercury, it has great relevance to toxicity on the kidneys, brain and liver tissues. The main nephrotoxic effects from exposure to mercury have been attributed to oxidative stress and production of reactive oxygen species (ROS), in addition to causing damage to mitochondrial metabolism by changing the functionality of the cell membrane and renal tubular polarity. The organic Hg forms present toxic effects on the central nervous system (CNS), with the ability to induce serious damage in the neurological deficiencies such as in speech, hyperactivity, difficulty concentrating and even disorder similar to autism. In liver tissue, the major toxic effects occur by changes in the enterohepatic cycle, key process excretion of mercury. Exposure to metals, in general, activates a mechanism which changes the production of proteins in an attempt to maintain homeostasis and to protect or repair macromolecules that are direct targets or indirect xenobiotics. In this sense, the aim of this study was to evaluate, by means of two-dimensional maps, protein expression\'profiles in liver, brain and kidney samples of rats exposed to different species of mercury (inorganic, methylmercury and ethylmercury) compared to their respective controls, using the classic platform for proteomic analysis (2DE and MS). It was observed that exposure to different forms of Hg modified expression of 139 proteins, 22 in brain cells 19 in kidney tissue cells, and 27 in the hepatic tissue cells. Among the differentially expressed proteins, they stood out to those directly related to oxidative stress in the liver and kidney tissues and related to homeostasis of Ca2+ and glutamate regulation in the synaptic cleft, the brain tissue. This study demonstrated that all Hg forms modified the protein profile in all tissues, and many altered proteins, corroborate the Hg mechanisms of action, as provided in the literature.

O mercúrio (Hg) é um metal não essencial, encontrado em diferentes formas químicas, com propriedades bastante diferentes. Entretanto, todas são tóxicas e capazes de alterar inúmeras vias metabólicas. Dentre os efeitos tóxicos da exposição ao Hg, tem grande relevância a toxicidade sobre os tecidos renal, cerebral e hepático. Os principais efeitos nefrotóxicos decorrentes da exposição ao Hg têm sido atribuídos ao estresse oxidativo e produção de espécies reativas do oxigênio (ROS), além de causar danos no metabolismo mitocondrial, alterando a funcionalidade da membrana celular e a polaridade tubular renal. As formas orgânicas do Hg apresentam efeitos tóxicos sobre o sistema nervoso central (SNC), com a capacidade de induzir sérios danos ao desenvolvimento neurológico, tais como deficiências na fala, hiperatividade, dificuldade de concentração e até mesmo desordens semelhantes ao autismo. No tecido hepático, os principais efeitos tóxicos ocorrem por alterações no ciclo entero-hepático, processo-chave na excreção do mercúrio. A exposição aos metais, de uma maneira geral, ativa um mecanismo que modifica a produção alterada de proteínas na tentativa de manter a homeostase e de proteger ou reparar macromoléculas que são alvos diretos ou indiretos de xenobióticos. Neste sentido, o objetivo deste trabalho foi avaliar, por meio de mapas bidimensionais, o perfil de expressão de proteínas em amostras fígado, cérebro e rim de ratos expostos a diferentes espécies de mercúrio (inorgânico, metilmercúrio e etilmercúrio) comparados aos seus respectivos controles, utilizando a plataforma clássica de análise proteômica (2DE e MS). Observou-se que a exposição às diferentes formas de Hg modificou a expressão de 139 proteínas, sendo 22 em células cerebrais, 19 em células do tecido renal, e 27 em células do tecido hepático. Dentre as proteínas diferencialmente expressas, destacaram-se àquelas relacionadas diretamente ao estresse oxidativo, nos tecidos hepático e renal e relacionadas à homeostase do Ca+2 e à regulação de glutamato na fenda sináptica, no tecido cerebral. Este estudo demonstrou que todas as formas de Hg, promoveram mudanças no perfil protéico em todos os tecidos e, muitas proteínas alteradas, corroboram com os mecanismos de ação do Hg, já previstos na literatura / Mercury (Hg) is not essential metal found in various chemical species with different properties, but all are toxic and can alter numerous metabolic pathways. Among the toxic effects of exposure to mercury, it has great relevance to toxicity on the kidneys, brain and liver tissues. The main nephrotoxic effects from exposure to mercury have been attributed to oxidative stress and production of reactive oxygen species (ROS), in addition to causing damage to mitochondrial metabolism by changing the functionality of the cell membrane and renal tubular polarity. The organic Hg forms present toxic effects on the central nervous system (CNS), with the ability to induce serious damage in the neurological deficiencies such as in speech, hyperactivity, difficulty concentrating and even disorder similar to autism. In liver tissue, the major toxic effects occur by changes in the enterohepatic cycle, key process excretion of mercury. Exposure to metals, in general, activates a mechanism which changes the production of proteins in an attempt to maintain homeostasis and to protect or repair macromolecules that are direct targets or indirect xenobiotics. In this sense, the aim of this study was to evaluate, by means of two-dimensional maps, protein expression\'profiles in liver, brain and kidney samples of rats exposed to different species of mercury (inorganic, methylmercury and ethylmercury) compared to their respective controls, using the classic platform for proteomic analysis (2DE and MS). It was observed that exposure to different forms of Hg modified expression of 139 proteins, 22 in brain cells 19 in kidney tissue cells, and 27 in the hepatic tissue cells. Among the differentially expressed proteins, they stood out to those directly related to oxidative stress in the liver and kidney tissues and related to homeostasis of Ca2+ and glutamate regulation in the synaptic cleft, the brain tissue. This study demonstrated that all Hg forms modified the protein profile in all tissues, and many altered proteins, corroborate the Hg mechanisms of action, as provided in the literature.

Host defense peptides (HDPs) provide innate immune defense against invasive S. aureus infection. Recent studies suggest potential cross-resistance between HDPs and the lipopeptide antibiotic, daptomycin (DAP). Isolates that exhibit DAP non-susceptible phenotypes may have virulence advantages and pose challenges to effective treatment. The current studies were performed to compare the efficacies and mechanisms of action of native and engineered HDPs vs. clinical S. aureus strain pairs which differed in susceptibility to daptomycin in vitro. Ultrasensitive radial diffusion and multi-colored flow cytometry were employed to analyze distinctive susceptibilities and mechanisms of resistance, respectively. Overall efficacies were greater vs. DAP-susceptible (DSSA) vs. DAP non-susceptible (DNSA) S. aureus isolates for some but not all HDPs. Efficacy profiles of certain HDPs were influenced by pH, regardless of whether the particular isolate was DSSA or DNSA phenotype. Mechanistically, DSSA and DNSA isolates differed in responses to specific HDPs regarding cell energetics, membrane permeability, cytoplasm membrane turnover, and cell death protease induction. DSSA and DNSA strain pairs exhibited non-identical mechanisms of resistance to HDPs. At pH 7.5, as expected, HDPs hNP-1 and RP-1 exerted significantly greater efficacy on susceptible control strain ISP479C vs. its resistant counterpart ISP479R. These data suggest different mechanisms of HDP resistance are active in differing DNSA strains. These preliminary results are under further investigation, as are the genetic determinant(s) that may emerge during infection. If substantiated, these findings would imply multiple modes of survival of S. aureus in the face of DAP or HDPs.

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No. of bitstreams: 2 license_rdf: 9 bytes, checksum: 42dd12a06de379d3ffa39b67dc9c7aff (MD5) isabela_pereira_gomes.pdf: 4600043 bytes, checksum: 3419d89b2ca19457442da4e144781e1b (MD5) Previous issue date: 2016 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Funda??o de Amparo ? Pesquisa do Estado de Minas Gerais (FAPEMIG) / Micro-organismos resistentes a antibi?ticos v?m atingindo ?ndices elevados nos ?ltimos anos, agravando problemas de sa?de p?blica e econ?micos. O impacto na sociedade devido ? resist?ncia de bact?rias aos antibi?ticos convencionais tem mobilizado a pesquisa para o desenvolvimento de novas drogas. Nesse contexto, surgem os pept?deos antimicrobianos, que s?o mol?culas efetoras do sistema imune inato e podem ser encontrados em praticamente todos os seres vivos. Para prote??o contra os micro-organismos presentes em seu habitat, a pele de anuros apresenta um verdadeiro arsenal qu?mico, constitu?do, em sua maioria, de diferentes classes de pept?deos. Dentre esses, os antimicrobianos s?o considerados os mais avan?ados participantes do sistema imunol?gico. V?rios estudos sugerem que os pept?deos antimicrobianos podem atuar por diferentes mecanismo como miceliza??o, forma??o de poros na superf?cie da membrana ou, ainda, pelo ataque a algum alvo intracelular. Embora a maioria dos modelos existentes proponham que pept?deos antimicrobianos exercem suas atividades biol?gicas por meio da intera??o com a membrana bacteriana, o mecanismo de a??o dessas mol?culas ainda n?o ? completamente compreendido. Dessa forma, o presente estudo tem como objetivo obter informa??es estruturais, conformacionais e de intera??o em ambientes que mimetizam membranas biol?gicas, do pept?deo Hylaseptina P1 (HSP-1), composto por 14 res?duos de amino?cidos e isolado do anuro da esp?cie Hyla punctata visando o entendimento do seu mecanismo de a??o. Neste trabalho, o pept?deo HSP-1 foi sintetizado manualmente atrav?s da s?ntese de pept?deos em fase s?lida via estrat?gia Fmoc. As prefer?ncias conformacionais do pept?deo em meios micelares de dodecilsulfato de s?dio (SDS), dodecilfosfocolina (DPC) e ves?culas fosfolip?dicas de 1-palmitoil-2-oleilfosfatidilcolina (PC) e 1-palmitoil-2-oleoil-fosfatidilglicerol (PG) foram avaliadas por Dicro?smo Circular. Observou-se que em meio aquoso HSP-1 adota estrutura desenovelada, enquanto que, em meios micelares e ves?culas fosfolip?dicas, apresentou conforma??o ?-h?lice. A estrutura tridimensional foi estudada na presen?a de micelas de SDS e DPC por Espectroscopia de Resson?ncia Magn?tica Nuclear em solu??o, revelando que, em micelas de SDS o pept?deo HSP-1 exibe conforma??o helicoidal mais prolongada do que em micelas de DPC, no qual foi poss?vel verificar pequena dobra da h?lice na regi?o N-terminal da cadeia pept?dica, sugerindo maior inser??o do pept?deo em micelas zwiteri?nicas. O efeito da adi??o de pept?deo no tamanho e na carga superficial de ves?culas fosfolip?dicas de PC e PC:PG (3:1) foi investigado por medidas de espalhamento de luz din?mica e Potencial Zeta. Verificou-se que a adi??o de HSP-1 resulta no aumento do di?metro hidrodin?mico (?Dh ? 20 nm) e aumento do potencial Zeta (?? ? 15 mV) de ves?culas unilamelares (LUV?s) de PC:PG, indicando intera??o eletrost?tica com a superf?cie das ves?culas. Quando ves?culas de PC foram tituladas com HSP-1, foi observada maior altera??o no di?metro hidrodin?mico das LUVs (?Dh ? 22 nm), enquanto que n?o se observaram altera??es significativas nos valores de potencial Zeta (?? ? 0 mV), sendo indicativo de uma maior inser??o do pept?deo em membranas zwiteri?nicas. Conforme observado nos resultados de Calorimetria de Titula??o Isot?rmica, a intera??o com ves?culas zwiteri?nicas foi da ordem de 102 maior do que o valor encontrado para intera??o com ves?culas negativas. Contudo, os dados termodin?micos revelaram a predomin?ncia de intera??es eletrost?ticas de HSP-1 com ves?culas de PC:PG e hidrof?bicas com ves?culas de PG. Finalmente, a capacidade de forma??o de poros de HSP-1 foi examinada por meio de medidas de extravasamento de carboxifluoresce?na (CF) em ves?culas de PC e PC:PG. Em PC-LUVs observou-se uma maior intensidade de fluoresc?ncia em compara??o com PC:PG-LUVs, sugerindo uma maior atividade l?tica do pept?deo em ves?culas zwiteri?nicas. A porcentagem m?xima de CF liberada foi superior em LUVs-PC (65%) quando comparado a LUVs de PC:PG (50%), confirmando que HSP-1 tem maior capacidade de permeabiliza??o em meio zwiteri?nico. Os dados de extravasamento demostraram tamb?m menor cin?tica de libera??o de CF em PC-LUVs, condizente com um mecanismo no qual h? maior inser??o do pept?deo na interface da bicamada lip?dica, e maior cin?tica de libera??o de CF em PC:PG-LUVs, de acordo com um mecanismo tipo carpete, o qual envolve apenas intera??o superficial do pept?deo com a membrana. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Qu?mica, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2016. / The resistance of microorganisms to antibiotics have reached high levels in recent years, exacerbating public health and economic problems. The impact of bacterial resistance on the global society has mobilized the research community for the development and new drugs discovery. In this context, antimicrobial peptides appear as an alternative antibiotics class, which are effector molecules of the innate immune system in almost every living beings. In order to protect against microorganisms present in their habitat the skin of frogs presents a real chemical arsenal consisting mostly of different peptides classes, which are considered the most advanced participants of the immune system. Several studies suggest that antimicrobial peptides may act by different models such as micellization, pore formation in membrane surface or also intracellular target attack. Although most of models propose that antimicrobial peptides exert their biological activities by interacting with the bacterial membrane, the mechanism of action is not still fully understood. In order to study the action mechanism of antimicrobial peptide Hylaseptina P1 (HSP-1), composed of 14 amino acid residues and isolated from Hyla punctate species, this work presents conformational and thermodynamic analysis of peptide-membrane interaction in biomimetic environments. In this work, the HSP-1 peptide was synthesized manually by solid phase peptide synthesis using Fmoc strategy. The conformational preferences of peptide were evaluated by Circular dichroism in sodium dodecylsulfate (SDS) and dodecilfosfocolina (DPC) micellar media or 1-palmitoyl-2-oleilfosfatidilcolina (PC) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (PG) phospholipid vesicles. A random conformation was observed to HSP-1 in aqueous medium while in micellar or phospholipid vesicles media HSP-1 showed a predominant ?-helix conformation. The three dimensional structure were obtained by solution Nuclear Magnetic Resonance in the presence of SDS and DPC micelles, revealing that HSP-1 exhibits a longer helical conformation in SDS than DPC micelles. In zwiterionic medium could be verified a subtle bend at the N-terminus, suggesting a partial insertion of the peptide in DPC micelles. The effects of adding peptide in size or surface charge of PC and PC:PG phospholipid vesicles were investigated by Dynamic Light Scattering and Zeta Potential measurements. The addition of HSP-1 to PC:PG-LUVs results in increasing of both hydrodynamic diameter (?Dh ? 20 nm) and zeta potential (?? ? 15 mV), indicating an electrostatic interaction with the surface vesicles. On the other hand, when PC-LUVs were titrated with HSP-1 a greater change in hydrodynamic diameter (?Dh ? 22 nm), no significant variations were observed in superficial charge, indicating a partial insertion of the peptide in zwitterionic PC membranes. In addition, the thermodynamic studies carried out by isothermal titration calorimetry showed a peptide-membrane interaction approximately 102 higher with PC-LUVs when compared to negative PC:PG-LUVs. Nevertheless, whereas the greater enthalpic contribution in PC:PG-LUVs revealed the predominance of electrostatic interactions with HSP-1, in PC vesicles predominate hydrophobic interactions. Finally, the ability of poring formation of HSP-1 was examined by carboxyfluorescein (CF) release from PC and PC:PG vesicles. It was observed a higher fluorescence intensity in PC-LUVs compared to PC:PG-LUVs, suggesting a greater lytic activity of the peptide in zwiterionic vesicles. The maximum percentage of CF released was approximately 65% to PC-LUVs and 50% to PC:PG-LUVs, confirming a greater membrane permeabilization in zwiterionic medium. Furthermore, this study has also demonstrated a lesser kinetics of CF leakage in PC-LUVs, consistent with a mechanism in which there is greater insertion of the peptide in the lipid bilayer interface. On the other hand, the higher kinetics of CF leakage in PC:PG?LUVs is in accordance with a carpet-like mechanism, which involve only superficial interaction between peptide and membrane.

El fuego bacteriano, causado por Erwinia amylovora, es una enfermedad muy importante a nivel comercial y económico porque afecta a plantas de la familia de las rosáceas y es especialmente agresiva en manzano (Pyrus malus) y peral (Pyrus communis), así como en plantas ornamentales (Crataegus, Cotoneaster o Pyracantha). Esta enfermedad está distribuida por todo el mundo en zonas climáticas templadas de Amércia del Norte, Nueva Zelanda, Japón, Israel, Turquí y Europa. En España, el fuego bacteriano fue detectado por primera vez en 1995 en el norte del País (Euskadi) y más tarde en nuevos focos aparecidos en otras áreas. La enfermedad puede ser controlada comercialmente mediante la aplicación de pesticidas quimicos (derivados de cobre, antibioticos). Sin embargo, muchos de los productos químicos presentan baja actividad o causan fitotoxicidad, y la estreptomicina, el producto más eficaz, esta prohibido en muchos países, incluyendo España. Por tanto, en ausencia de apropiados agentes químicos, el control biológico se contempla como una buena alternativa. En el presente trabajo, un agente de control biológico, Pseudomonas fluorescens EPS62e, ha sido seleccionada de entre 600 aislados de las especies P. fluorescens y Pantoea agglomerans obtenidos de flores, frutos y hojas de plantas de la familia de las rosáceas durante una prospección llevada a cabo en varias áreas geográficas de España. La cepa ha sido seleccionada por su capacidad de suprimir la infecciones producidas por E. amylovora frutos inmaduros, flores y brotes de peral en condiciones de ambiente controlado, presentando unos niveles de control similares a los obtenidos mediante el control químico usando derivados de cobre o antibióticos. La cepa además ha mostrado la capacidad de colonizar y sobrevivir en flores y heridas producidas en frutos inmaduros en condiciones de ambiento controlado pero también en flores en condiciones de campo. La exclusión de E. amylovora medinate la colonización de la superficie, el consumo de nutrientes, y la interacción entre las células del patógeno y del agente de biocontrol es la principal causa de la inhibición del fuego bacteriano por la cepa EPS62e. Estas características constituyen aspectos interesantes para un desarrollo efectivo de la cepa EPS62e como un agente de biocontrol del fuego bacteriano en condiciones comerciales. / Fire blight, caused by Erwinia amylovora is a serious disease of rosaceous plants with great commercial and economic interest that is distributed over the world. Disease may be controlled commercially by the application of chemicals (copper compounds, antibiotics). Many chemical agents have low activity or cause phytotoxicity, and streptomycin, the most effective antibiotic, is not approved for use in many countries, including Spain. Therefore, in the absence of suitable chemical control agents, biological control could provide a useful alternative. In the present work, a biological control agent, Pseudomonas fluorescens EPS62e, has been selected among 600 isolates of P. fluorescens and Pantoea agglomerans obtained from flowers, fruits and leaves of rosaceous plants in a survey performed through several geographic areas of Spain. This strain has been selected for its capacity to suppress immature fruit, blossom and shoot infections caused by E. amylovora, under controlled environment conditions, providing control levels similar to chemical control with copper or antibiotic compounds. The strain has also shown the capacity to colonize and survive well in flowers and wounds on immature fruit under controlled environment conditions but also in flowers under natural conditions. Pre-emptive exclusion of the pathogen E. amylovora by surface colonization and nutrients depletion, and cell-to-cell interaction appear to be the main mechanisms of biocontrol. These characteristics constitute interesting traits for an effective development as a fire blight biological control agent under commercial conditions.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Not embargoed. Vita. Bibliography: 158-177.

Les nématodes gastro-intestinaux (NGI) représentent un problème sanitaire majeur dans le monde entier en systèmes de production à l’herbe des élevages de moutons et de chèvres. La maîtrise de ces parasites est désormais compliquée par l'apparition de résistances aux anthelminthiques (AH) chez les nématodes. L'utilisation de plantes riches en tanins (PRT) comme AH non conventionnels est une des alternatives possibles au contrôle chimique de ces maladies parasitaires. Cependant, le mécanisme d'action de ces plantes sur les vers adultes demeure inconnu. Cet objectif a constitué le premier but de cette étude en particulier en essayant de déterminer quelle part jouent les effets directs et indirects dans l’action des PRT sur les populations de Nématodes adultes dans le tractus digestif des petits ruminants. Un effet direct correspond à une action de type pharmacologique sur la biologie, la structure et l’ultrastructure des nématodes liés à des composés biochimiques présents dans les PRT. L'hypothèse d’un effet indirect correspondrait une modification de la réponse cellulaire dans les muqueuses gastro-intestinales de l'hôte, affectant la biologie des NGI. Cette thèse est divisée en deux parties: 1) Deux expériences in vivo réalisées respectivement, au Mexique et en France, visant à déterminer les effets directs ou indirects sur la biologie des NGI liés à la consommation de PRT tel le tzalam (Lysiloma latisiliquum), une Légumineuse arbustive du Yucatan, le sainfoin (Onobrychis viciifolia) une Légumineuse fourragère tempérée, et ainsi que le quebracho(Schinopsis spp.), un extrait riche en tannins condensés Les agneaux ont été infestés par Haemonchus contortus (au Mexique) ou H. contortus et Trichostrongylus colubriformis (en France). La consommation de tzalam a provoqué une perturbation de croissance des vers ou de fertilité des femelles d’H. contortus. Selon la durée de consommation, le quebracho a réduit la population de H. contortus et T. colubriformis, et affecte également la fertilité de T. colubriformis. Le sainfoin a affecté la fécondité de H. contortus sans affecter celle de T. colubriformis. Le comptage de cellules effectrices dans les muqueuses pour évaluer l’implication d’un effet indirect n'a pas montré de différences significatives entre les lots recevant ou non des PRT. La seconde partie s’est fondée sur un test in vitro et deux essais in vivo chez des chèvres infestées par H. contortus pour mieux comprendre l'effet de la consommation de PRT (tzalam, au Mexique ou sainfoin, en France) sur la structure et l’ultrastructure d’H. contortus. Les principales altérations ont été trouvées dans la cuticule et la région céphalique des vers. Des agrégats de matériel végétal autour de la vulve n’ont été trouvé qu’in vitro. L’examen de l’ultrastructure des H. contortus a montré des signes de vacuolisation, surtout visible dans les tissus intestinaux et musculaires. Ces lésions suggèrent que des composés secondaires présents dans le feuillage des PRT peuvent être impliqués dans les fonctions vitales d’ H. contortus, telles que la mobilité, la nutrition et éventuellement la reproduction. / Gastrointestinal nematodes (GIN) represent a major health problem worldwide in grazing sheep and goat production systems. The control of these parasites has been complicated by the emergence of nematodes which are resistant to the commercially available anthelmintics (AHs). The use of tannin rich plants (TRP), as non-conventional Ahs, represents an alternative for the control of these parasites. However, the mechanism of action of such plants against adult populations of GIN has not been determined. The objective of the study was to determine the direct and indirect effect of TRP against adult populations of GIN in small ruminants. A direct effect is considered to be any action against the biology, structure and/or ultrastructure of the nematodes which is similar to a chemical AH. The indirect effect is a modification of the immune response of the host at the level of the gastrointestinal mucosa which affects the biology of the GIN. This study is divided into two stages: First stage) Two in vivo experiments conducted in Mexico and France respectively, determined the direct and indirect effects on the biology of GIN due to the consumption of the TRP forage of tzalam (Lysiloma latisiliquum), sainfoin (Onobrychis viciifolia) as well as a tannin-rich quebracho extract (Schinopsis spp). Lambs were artificially infected with Haemonchus contortus (Mexico) or H. contortus and Trichostrongylus colubriformis (France). The consumption of tzalam affected the length and fertility of H. contortus females. Meanwhile, the quebracho extract reduced the population of H. contortus and T. colubriformis also affecting the fecundity of T. colubriformis. The sainfoin affected the fecundity of H. contortus without affecting T. colubriformis. The indirect effect was not evident. Second stage) An in vitro assay and two in vivo experiments with goats infected with H. contortus allowed to identify the effect of the consumption of TRP (tzalam or sainfoin) on the structure and ultrastructure of H. contortus. Alterations in cuticle and cephalic region were found. The aggregates in the vulva were only found in vitro. The ultrastructure of H. contortus showed a vacuolization process in the intestinal and muscular tissues of these nematodes. The lesions suggest that the compounds contained in the foliage of TRP may be involved in vital functions of H. contortus such as mobility, nutrition and possibly reproduction. This study is a contribution towards the understanding the mechanisms of AH action of the TRP against GIN.

La soca EPS125 ha mostrat ser un efectiu agent de control biològic de diferents patògens fúngics de postcollita en diferents fruits. Degut a la seva elevada eficàcia, es va plantejar desenvolupar aquesta soca comercialment i per aquest motiu en el present treball es plantejà complementar la informació necessària pel seu registre. D'acord amb els resultats obtinguts mitjançant proves fenotípiques i genotípiques, la soca EPS125 queda inclosa dins l'espècie Pantoea agglomerans (Enterobacter agglomerans-Erwinia herbicola). En relació a la utilització de fonts de carboni, en el perfil i contingut d'àcids grassos cel·lulars i en el polimorfisme en la longitud dels fragments de macrorestricció genòmica (MRFLP), la soca EPS125 mostrà trets característics que la diferencien d'altres soques. Els dos marcadors moleculars (125.2 i 125.3) específics per la soca EPS125 dissenyats en el present treball mostraren ser semiespecífics per la seva detecció mitjançant la tècnica PCR i Real Time PCR. Quedant pendent l'anàlisi d'especificitat de l'ús combinat dels dos marcadors moleculars en una reacció PCR multiplex. P. agglomerans EPS125 ha mostrat ser molt efectiva en el control de Penicillium expansum en poma amb una dosi efectiva mitjana de 2.7x105 a 7x105 ufc/ml, i una ratio de 25-101 cèl·lules de la soca EPS125 per inactivar una espora del patogen segons el model de saturació hiperbòlica. Segons les aproximacions fenotípiques i estudis genotípics realitzats, sembla que els mecanismes de biocontrol utilitzats per la soca EPS125 contra P. expansum en poma estan directament relacionats amb la capacitat de formació de biofilm per aquesta soca. / Strain EPS125 has shown effectiveness against a wide range of fungal pathogens in a large variety of fruit. However, to develop this strain as commercial biopesticide an extensive characterization is essential. For this reason, the objective of this PhD thesis was to complete the necessary information for its future registration.According to morphological and biochemical tests, strain EPS125 pertain to Pantoea agglomerans (Enterobacter agglomerans-Erwinia herbicola) species. This strain showed typical traits different from other bacteria in relation to the ability to use several carbon sources, the fatty acid profiles and the macrorestriction fragment length polymorphism (MRFLP) pattern. The two DNA molecular markers of P. agglomerans EPS125 (125.2 and 125.3) obtained in the present work were semispecific in the detection of strain EPS125 by means of PCR and Real Time PCR. However, the combined use of the two primer sets in a multiplex PCR reaction would be specific. P. agglomerans EPS125 was highly effective against P. expansum in apple fruit having a median effective dose from 2.7x105 to 7x105 cfu/ml and a ratio of 101 and 25 EPS125 cells to inactivate one pathogen spore according to the hyperbolic saturation model. Biocontrol mechanisms used by P. agglomerans EPS125 against P. expansum in apple fruit may be related with the ability of biofilm formation by this strain as show phenotypic approaches and genotypic studies.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The intake of flavonoids is closely associated with diminished risks of cancer, atherosclerosis and other chronic inflammation disturbance related. These compounds are described, mainly, by their anti-inflammatory and antioxidant activities. Myricitrin is a flavonoid that inhibits protein kinases and NO production. Therefore, the research of the mechanisms by which these compounds exerts their effects is extremely interesting for the therapeutic application. In this study, the antinociceptive and anti-inflammatory activities of myricitrin as well as its mechanisms of action were investigated. The systemic (p.o. or i.p.) and central (i.c.v. or i.t.) administration of myricitrin reduced in a dose-dependent manner the visceral pain induced by acetic acid. The i.p. treatment with myricitrin also prevented nociception induced by i.pl. injection of glutamate, capsaicin, PMA and by i.t. injection of glutamate, SP, TNF-α and IL-1β in mice. In addition, myricitrin treatment inhibited mechanical hyperalgesia induced by BK, but not that induced by epinephrine or PGE2 in rats. Western blot analysis revealed that myricitrin treatment fully prevented PKCα and PKCε activation by PMA in mice hindpaw. The antinociception caused by myricitrin in the acetic acid test was significantly reverted by Gi/o protein inactivation (pertussis toxin treatment); treatment with ATPgated K+ channel blocker (glibenclamide), CaCl2 and L-arginine (NO precursor) administration. However, myricitrin-induced antinociception was modified neither by antagonist opioid (naloxone) nor by neonatal capsaicin treatment (which depletes 80% of unmyelinated C fibers). In addition, myricitrin effects were not associated with sedative and muscle-relaxant action. In vitro assays using slices of cerebral cortex of rats revealed that myricitrin inhibited calcium transport in a depolarizing condition; however, at higher concentration, it inhibited calcium transport also in a non-depolarizing condition. Myricitrin increased nociceptive threshold in mechanical allodynia induced by both partial sciatic nerve ligation (neuropathic pain) and FCA i.pl. injection (inflammatory chronic pain). This same treatment decreased paw edema, morphological alterations and MPO activity (proinflammatory enzyme) in FCA hindpaw. On the other hand, it did not reduce neutrophils migration to inflammation site. Myricitrin produced potent antioxidant activity when assessed by Fe2+-induced lipid peroxidation. In conclusion, the present study showed that myricitrin exhibits antinociceptive and anti-inflammatory activity when analyzed in acute and chronic models of nociception. The mechanisms involved in the myricitrin beneficial effects included Gi/o protein activation, ATP- gated K+ channels opening, inhibition of PKC, NO synthesis, wedged of Ca2+ transport, inhibition of MPO activity and scavenger action. / O consumo de flavonóides através da dieta está positivamente relacionado com a diminuição do risco de câncer, ateroesclerose e agravamento de doenças relacionadas com inflamação e estresse oxidativo. Estes compostos são descritos, principlamente, pelas suas ações antiinflamatórias e antioxidantes. A miricitrina é um flavonóide que possui propriedades de inibir proteínas quinases, a síntese de NO entre outras. Desta forma, é de grande interesse a pesquisa destes compostos com finalidade terapêutica. No presente trabalho investigou-se as propriedades antinociceptivas e antiinflamatórias do flavonóide miricitrina, bem como os possíveis mecanismos envolvidos em tal processo. A administração sistêmica (oral ou i.p.) e central (i.c.v. ou i.t.) de miricitrina reduziu de forma dependente da dose o número de contorções abdominais induzidas pelo ácido acético. O tratamento i.p. com a miricitrina também preveniu a nocicepção induzida pela injeção i.pl. de glutamato, capsaicina e PMA, bem como, a nocicepção induzida pela injeção i.t. de glutamato, SP, TNF-α e IL-1β em camundongos. Além disso, o tratamento com miricitrina inibiu a hiperalgesia mecânica induzida pela BK, mas não aquela induzida pela epinefrina e PGE2 em ratos. Análises de Western blot revelaram que o tratamento com miricitrina inibiu completamente a ativação da PKCα e PKCε induzida pela injeção i.pl. de PMA. A antinocicepção causada pela miricitrina no teste do ácido acético foi significativamente revertida pelo pré-tratamento dos animais com o inativador da proteína Gi/o (toxina pertussis); com o bloqueador de canal de K+ (glibenclamida); com o precursor de NO (L-arginina) e o CaCl2. Entretanto, a antinocicepção provocada pela miricitrina não foi afetada pelo tratamento com antagonista opióide (naloxona); pelo tratamento neonatal com capsaicina (destrói 80% das fibras sensorias não mielinizadas do tipo C) e não está relacionada com uma ação sedativa, relaxante muscular ou hipotérmica do composto. Além disso, ensaios in vitro em fatias de córtex cerebral de ratos revelaram que a miricitrina inibe o transporte de cálcio em uma condição despolarizante, embora, quando em alta concentração, miricitrina inibe o transporte de cálcio também em condição nãodespolarizante. A miricitrina reduziu a alodínia mecânica induzida pela ligadura parcial de nervo ciático (dor neuropática) e pela injeção i.pl. de FCA (dor inflamatória crônica). Este mesmo tratamento reduziu o edema de pata, as alterações morfológicas e a atividade da MPO (enzima pró-inflamatória) na pata injetada com FCA, porém não reduziu a migração de neutrófilos ao local da inflamação. A miricitrina mostrou potente ação antioxidante frente à peroxidação lipídica induzida por Fe2+. De acordo com o presente trabalho pode-se concluir que a miricitrina é dotada de atividade antinociceptiva e antiinflamatória quando avaliada em modelos de nocicepção aguda e crônica. Os mecanismos de sua ação antinociceptiva e antiinflamatória incluem a ativação de proteína Gi/o e abertura de canais de K+, a inibição da PKC, da síntese de NO, bloqueio do transporte de Ca2+, inibição da atividade da MPO e neutralização de radicais livres.

This thesis is focused on the issue of violence in civil society. Using Charles Tilly's political theory, it attempts to point out that the emergence of collective violence can be well understood by tracing small scale causes (mechanisms), rather than large causes (poverty, extremism etc.). This argument is empirically studied in the context of anti-Roma riots that took place in Šluknov Hook, Czech republic, in the year 2011. The research is based on a broad concept of civil society, which doesn't assume fixed division between civil and uncivil subjects, but works with a number of actors, whose identity is unstable and their acting strategy fluently changes from nonviolent to violent and contra. The data show, that radical actors are generally more prone to use force. However, Tilly's theory provides opportunity to explain their influence on majority through the dynamics of relationships that is studied in this paper. The catalogue of events was created on the basis of news and document analysis and the incidence of theoretically defined mechanisms is identified by process tracing method: Boundary activation between us and them (mainly network-based escalation, signaling spirals), polarization, competitive display, selective retaliation, containment, monitoring, certification/decertification and...