Novas alternativas de produção de ácidos graxos altamente insaturados n-3 (HUFA n-3) são procuradas mediante várias abordagens experimentais na área aquícola considerando que a oferta atual de óleos marinhos é limitada. Como os ácidos graxos das séries n-3 e n-6 são metabolizados mediante os mesmos processos enzimáticos, o aporte de ácidos graxos essenciais da dieta poderia influenciar nos níveis de alongamento e desaturação em função da disponibilidade relativa destes compostos. O objetivo deste trabalho foi determinar a influência de diferentes relações 18:3n3/18:2n6 (ALA/LA - ácido linolênico/ácido linoleico) de rações exclusivamente vegetais, sobre o metabolismo de ácidos graxos de juvenis de pacu (Piaractus mesopotamicus). Inicialmente 18 unidades experimentais (caixas com 16 peixes) foram alimentadas por 34 dias (C1) com uma dieta controle (tratamento T6) contendo óleo de tilápia como único ingrediente de origem animal. Posteriormente, foram formados aleatoriamente, 6 grupos com 3 unidades experimentais cada. Em uma segunda fase, durante 72 dias, 5 grupos receberam dietas, contendo óleo de linhaça e soja em substituição ao óleo de tilápia, com relações calculadas de ALA/LA de: 2,98 (T1); 1,68 (T2); 1,03 (T3); 0,61 (T4) e 0,35 (T5) e um grupo continuou com a dieta controle (T6). Nesta segunda fase, determinaram-se parâmetros de desempenho zootécnico e metabolismo de ácidos graxos em 3 intervalos de 24 dias (C2=58 dias, C3=82 dias e C4=106 dias). Os animais apresentaram crescimento e ganho de peso sem diferenças significativas entre os grupos e com aumento progressivo da proporção de gordura corporal total. A composição de ácidos graxos da dieta influenciou de forma proporcional sobre a composição da maioria de ácidos graxos de corpo inteiro. Os coeficientes de digestibilidade aparente aumentaram conforme o grau de instauração dos ácidos graxos e sua abundância relativa na dieta. A concentração de ácidos graxos polinsaturados nos lipídios de corpo inteiro não diferiu entre a maioria de tratamentos. O teor e aparecimento do ácido graxo 20:5n3 (EPA) e a atividade da enzima Δ-5 desaturase dos grupos T1, T2 e T3 foi menor que os demais tratamentos no período C4. O aparecimento de HUFA n-3 e n-6, atividade Δ-6 desaturase total e atividade Δ-6 desaturase sobre os ácidos graxos n-3 totais dos tratamentos T1 a T5 foram maiores que no T6 (controle). Estes resultados demonstram que o pacu (Piaractus mesopotamicus) é capaz de alongar e desaturar ácidos graxos essenciais para produzir ácidos graxos altamente insaturados (ácidos graxos com no mínimo 20 C e 3 ligações duplas - HUFA). Esta atividade é diminuída pela presença, mesmo em quantidades pequenas, de HUFA na dieta. Além disso, sugerem que relações ALA/LA da dieta, iguais ou maiores que 1, provocam diminuição na taxa de produção de 20:5n3, nos níveis testados neste estudo. / New alternatives for production of highly unsaturated fatty acids n-3 (n-3 HUFA) are sought by various experimental approaches in aquaculture, considering that the current supply of marine oils is limited. Since fatty acids of the n-3 and n-6 series are metabolized by the same enzyme processes, the supply of essential fatty acids of the diet could influence the elongation and desaturation levels, depending on the relative availability of these compounds. The objective of this study was to determine the influence of different 18:3n-3/18:2n-6 ratios (ALA/LA - linolenic acid/linoleic acid) of diets containing exclusively vegetable ingredients on the metabolism of fatty acids, of pacu juveniles (Piaractus mesopotamicus). Initially, 18 experimental units (boxes with 16 animals) were fed a control diet (T6 treatment) containing tilapia oil as the sole animal ingredient for 34 days (C1). Later, 6 groups with 3 experimental units each were formed at random. In a second phase, during 72 days, five groups were fed diets containing linseed and soy oils to replace tilapia oil, with calculated ALA/LA ratios: 2.98 (T1); 1.68 (T2); 1.03 (T3); 0.61 (T4) and 0.35 (T5) and a group continued with the control diet (T6). In the second phase were determined growth performance parameters and fatty acid metabolism in three intervals of 24 days (C2 = 58 days = 82 days and C3 C4 = 106 days). The animals showed growth and weight gain with no significant differences between groups and progressive increase in the proportion of total body fat. The fatty acid composition of the diet influenced proportionally on the composition of the majority of whole body fatty acids. The apparent digestibility increased as the degree of unsaturation of fatty acids and their relative abundance in the diet. The concentration of polyunsaturated fatty acids in whole body lipids did not differ among the most treatments. The content and appearance of the fatty acid 20:5n3 (EPA) and the activity of Δ-5 desaturase enzyme of groups T1, T2 and T3 was lower than the other treatments in C4 period. The appearance of n-3 and n-6 HUFA, Δ 6-desaturase activity and the total Δ 6-desaturase activity on the total n-3 fatty acids from T1 to T5 treatments were higher than T6 (control). These results demonstrate that pacu juveniles are able to elongate and desaturate essential fatty acids to produce highly unsaturated fatty acids. This activity diminishes by the presence of even small amounts of HUFAs in the diet. Moreover, it suggests that ALA/LA ratio of diet, equal to, or greater than one, cause a decrease in the rate of production of 20:5n3, within the levels tested in this study.

A selenocisteína (Sec, U) é o aminoácido que representa a principal forma biológica do elemento selênio e sua incorporação é um processo co-traducional em selenoproteínas como resposta ao códon UGA em fase e requer uma complexa maquinaria molecular. O repertório completo de genes envolvidos nessa via de síntese em procariotos é conhecido, porém algumas das interações moleculares ainda não foram totalmente esclarecidas. Este projeto visa à caracterização molecular nas interações entre o Fator de Elongação específico para incorporação de Sec (SelB) e Seril-tRNA sintetase (SerRS) com distintas construções do tRNASec de Escherichia coli afim de compreender a sua especificidade, seletividade e ordem de eventos. Para isso, medidas de Espectroscopia de Anisotropia de Fluorescência (FAS), Ultracentrifugação Analítica (AUC) e Calorimetria de Varredura Diferencial (DSC) foram utilizadas para determinação das constantes de interação desses complexos proteína-tRNA. Além disto, experimentos de Espalhamento de Raios-X a baixo ângulo (SAXS) e Microscopia eletrônica de transmissão por contraste negativo (NS-EM) foram realizados para elucidação estrutural destes complexos. Os estudos propostos irão auxiliar no entendimento do mecanismo de incorporação e de especificidade do tRNA para este aminoácido em bactérias bem como nos demais domínios da vida além de possibilitar um aumento na compreensão de complexos do tipo proteína-tRNA bem como salientar a importância dos elementos estruturais do tRNA para sua especificidade no processo de síntese de novas proteínas. / Selenocysteine (Sec, U) is an amino acid that represents the main biological form of the selenium element and its incorporation is a co-translational process in selenoproteins in response to the in-phase UGA codon and requires complex molecular machinery. The complete repertoire of genes involved in this pathway of synthesis in prokaryotes is known, although some of the molecular interactions have not yet been fully elucidated. This project aims at the molecular characterization in the interactions between the specific elongation factor for the incorporation of Sec (SelB) and Seril-tRNA synthase (SerRS) with different constructions of tRNASec from Escherichia coli in order to their specificity, selectivity and order of events. For this, measurements using Fluorescence Anisotropy Spectroscopy (FAS), Analytical Ultracentrifugation (AUC) and Differential Scanning Calorimetry (DSC) were employed to determine the interaction constants of the protein-tRNA complexes. In addition, Small Angle X-Ray Scattering (SAXS) experiments and negative stain transmission electron microscopy (NS-EM) were performed for structural elucidation of these complexes. The proposed studies will help to understand the mechanism of tRNA incorporation and specificity for this amino acid in bacteria as well as other domains of life. In addition, it allows an increase in the understanding of protein-tRNA-like complexes as well as emphasizing the importance of structural elements of tRNA for its specificity in the process of synthesis of new proteins.

O experimento foi conduzido na FZEA/USP – Pirassununga/SP de dezembro de 2002 a março de 2003, em uma área de 26,7 ha formada de capim-braquiarão. O objetivo foi avaliar as características morfofisiológicas durante a fase recuperação da planta forrageira após o pastejo. A pastagem foi submetida a quatro intensidades de pastejo representadas por níveis de oferta de forragem (5, 10, 15 e 20% - kg MS/100 kg peso animal.dia), utilizando-se o método de lotação rotacionada, sendo sete dias de ocupação e vinte e oito dias de descanso. O delineamento experimental foi em blocos completos e casualizados, com quatro repetições. Foram avaliadas as seguintes respostas: (1) características morfogênicas: intervalo de aparecimento foliar (IApF), taxa de aparecimento foliar (TApF), taxa de alongamento foliar (TAlF), taxa de senescência foliar (TSenF), taxa de alongamento de colmo (TAlC); e (2) características estruturais: número de folhas vivas por perfilho (NFVP), comprimento de folhas verdes por perfilhos (CFVP), comprimento médio de folhas verdes por perfilho (CMFVP) e comprimento de colmo (CC), dinâmica de perfilhamento, englobando, percentagem de perfilhos decapitados (PPD), número de perfilhos basais remanescentes (NPBR), número de perfilhos aéreos remanescentes (NPAR), número de perfilhos basais novos (NPBN), número de perfilhos aéreos novos (NPAN), densidade populacional de perfilhos (DPP), peso médio de perfilho seco, (PMPS) e peso médio de perfilho verde (PMVP). Pastagens submetidas a menor intensidade de pastejo apresentam maior TSenF. As condições climáticas que favorecem o processo de florescimento foram determinantes nessa TSenF. O avanço na estação de crescimento interferiu de forma negativa na TApF, aumentando automaticamente o IApF. A TAlF teve pouca influência dos tratamentos de OF, exceto na condição inicial de manejo, período de avaliação 1 (PAv.1). Nos PAv. o aumento do NFVP esteve relacionado com a diminuição de seu comprimento médio. O CC aumentou com o avançar dos PAv. A maior intensidade de pastejo condicionou o aumento na PPD, a maior DPP e o menor PMPS e PMPV. As condições climáticas tiveram papel importante na sobrevivência e aumento dos NPBR nos PAv.. A sobrevivência dos NPAR esteve condicionada aos efeitos da OF. Condições de maior intensidade de pastejo favoreceu o aparecimento de NPBN e NPAN. No avançar dos PAv., o pastejo leniente provocou diminuição do surgimento do NPAN, embora tenham aumentado durante o período de rebrotação. / The experiment was conducted in the FZEA/USP - Pirassununga/SP from December/2002 to March/2003, in a pasture of signal grass of 26.7 ha. The objective was evaluated the morphophysiological characteristics during regrowth of the plant after grazing. The pasture was submitted at grazing intensities represented by four levels of herbage allowance (5, 10, 15 and 20% - kg DM/100 kg weight animal.day), using rotational stocking method, being seven days of occupation and twenty and eight days of interval of grazing. The experimental design was in complete randomised block, with four replications. The evaluated responses were: (1) morphogenetics characteristics: leaf appearance interval (LApI), leaf appearance rate (LApR), leaf elongation rate (LElR), leaf senescence rate (LSenR), stem elongation rate (SElR); e (2) structural characteristics: number of live leaves (NLL), length of green leaf (LGL), length average green leaf (LAGL) and length of stems (LS), dynamics of tillering, percentage of decapitated tiller (PDT), number of remaining basal tiller (NRBT), number of remaining aerial tiller (NRAT), number of new basal tiller (NNBT), number of new aerial tiller (NNAT), population density of tiller (PDT), dry average weight for tiller, (DAWT) and green average weight for tiller (GAWT). Pasture submitted to lenient grazing presented higher LSenR. The climatic conditions that favour flowering process were determinant in LSenR. The advance in growth station influenced negatively in the LApR and increased automatically the LApI. The HA had little influence on the LAIR, except in the initial condition of management, period of evaluation 1 (PAv.1). In the PAv. the increase of the NLL was related with the reduction of its average length. The LS increased with advancing of the PAv.. The highest grazing intensity conditioned the increase in the PDT, the biggest PDT and lowest DAWT and GAWT. The climatic conditions were important in the survival and increase of the NRBT in the PAv.. The survival of the NRAT was conditioned to the effect of the HA. The highest grazing intensity favoured the appearance of NNBT and NNAT. With the advance of the PAv., the lenient grazing provoked reduction of the NNAT emergence, even so has increased during regrowth period.

Chu Lai On. / Thesis submitted in: July 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 152-172). / Abstracts in English and Chinese. / Acknowledgements --- p.2 / Abstract --- p.3 / Table of Content --- p.7 / Abbreviations --- p.12 / Naming system for mutant proteins --- p.13 / Abbreviations for amino acid --- p.14 / Chapter Chapter 1 --- Introduction --- p.15 / Chapter 1.1 --- Structure-function relationship of trichosanthin --- p.18 / Chapter 1.2 --- Properties of acidic ribosomal P-proteins --- p.21 / Chapter 1.3 --- Interaction among P-proteins and trichosanthin --- p.25 / Chapter 1.4 --- Properties of eukaryotic elongation factor 2 and interaction with P-proteins --- p.26 / Chapter 1.5 --- "Objectives and strategy of studying the interaction among trichosanthin, P-proteins and eukaryotic elongation 2" --- p.30 / Chapter Chapter 2 --- Materials and Methods --- p.33 / Chapter 2.1 --- General techniques --- p.33 / Chapter 2.1.1 --- Preparation and transformation of Escherichia coli competent cells --- p.33 / Chapter 2.1.2 --- Minipreparation of plasmid DNA using Wizard Plus SV Minipreps DNA purification kit from Promega --- p.34 / Chapter 2.1.3 --- Agarose gel electrophoresis of DNA --- p.36 / Chapter 2.1.4 --- Purification of DNA from agarose gel using Wizard SV Gel and PCR Clean-Up System from Promega --- p.36 / Chapter 2.1.5 --- Polymerase Chain Reaction (PCR) --- p.37 / Chapter 2.1.5.1 --- Basic Protocol --- p.37 / Chapter 2.1.5.2 --- Generation of P2 truncation mutants --- p.38 / Chapter 2.1.5.3 --- Generation of TCS mutants --- p.39 / Chapter 2.1.6 --- Restriction digestion of DNA --- p.41 / Chapter 2.1.7 --- Ligation of DNA fragments --- p.41 / Chapter 2.1.8 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.42 / Chapter 2.1.9 --- Staining of protein in polyacrylamide gel --- p.45 / Chapter 2.2 --- Expression and purification of recombinant proteins --- p.46 / Chapter 2.2.1 --- "Bacterial culture, harvesting and lysis" --- p.46 / Chapter 2.2.2 --- Purification of recombinant TCS and mutants --- p.47 / Chapter 2.2.3 --- Purification of acidic ribosomal protein P2 and mutants --- p.48 / Chapter 2.2.4 --- Purification of MBP-fusion proteins --- p.50 / Chapter 2.3 --- Purification of eEF2 from rat livers --- p.51 / Chapter 2.4 --- In vitro binding assay by NHS-activated Sepharose resin --- p.53 / Chapter 2.4.1 --- Coupling of protein sample to NHS-activated Sepharose resin --- p.53 / Chapter 2.4.2 --- In vitro binding of protein sample to coupled NHS-activated resin --- p.54 / Chapter 2.5 --- Ribosome-inactivated activity assay using rabbit reticulocyte lysate in vitro translation system --- p.55 / Chapter 2.6 --- Circular dichroism (CD)spectrometry --- p.57 / Chapter 2.7 --- Isothermal titration calorimetry (ITC) experiment --- p.57 / Chapter 2.8 --- Surface plasmon resonance (SPR) experiment --- p.58 / Chapter 2.8.1 --- Immobilization of P2 onto aminosilane cuvette --- p.58 / Chapter 2.8.2 --- Interaction between eEF2 and immobilized P2 --- p.60 / Chapter 2.9 --- Preparation of Anti-P antibody --- p.61 / Chapter 2.10 --- Western blotting of protein --- p.62 / Chapter 2.11 --- Reagents and buffer --- p.64 / Chapter 2.11.1 --- Reagents for competent cell preparation --- p.64 / Chapter 2.11.2 --- Nucleic acids electrophoresis buffer --- p.65 / Chapter 2.11.3 --- Media for bacterial culture --- p.66 / Chapter 2.11.4 --- Buffers for TCS purification --- p.67 / Chapter 2.11.5 --- Buffers for eEF2 purification --- p.68 / Chapter 2.11.6 --- Reagents for SDS-PAGE --- p.68 / Chapter 2.11.7 --- Reagents and buffers for Western blot --- p.70 / Chapter 2.11.8 --- Reagents and buffers for coupling sample proteins to NHS-activated Sepharose resin --- p.72 / Chapter 2.11.9 --- Reagents and buffers for in vitro binding assay --- p.72 / Chapter 2.11.10 --- Reagents and Buffers for surface plasmon resonance --- p.72 / Chapter 2.12 --- Sequences of primers --- p.73 / Chapter Chapter 3 --- Interaction between TCS and P2 --- p.80 / Chapter 3.1 --- Introduction --- p.80 / Chapter 3.2 --- Interaction between TCS and P-proteins in rat liver lysate --- p.83 / Chapter 3.3 --- Construction of TCS mutants --- p.85 / Chapter 3.4 --- Expression and purification of TCS mutants --- p.87 / Chapter 3.5 --- Biological assay of TCS mutants --- p.91 / Chapter 3.6 --- Physical interaction of TCS mutants and P2 by surface plasmon resonance (SPR) --- p.94 / Chapter 3.7 --- Discussion --- p.100 / Chapter Chapter 4 --- Mapping the region of P2 that binds TCS and eEF2 --- p.104 / Chapter 4.1 --- Introduction --- p.104 / Chapter 4.2 --- Construction of P2 truncation mutants --- p.106 / Chapter 4.3 --- Expression and purification of P2 truncation mutants --- p.107 / Chapter 4.4 --- Mapping the region of P2 that binds TCS --- p.111 / Chapter 4.4.1 --- Interaction between TCS and P2 mutants by in vitro binding assay --- p.111 / Chapter 4.4.2 --- Interaction study of TCS and P2 mutant by isothermal titration calorimetry (ITC) --- p.116 / Chapter 4.5 --- Mapping the region of P2 that binds eEF2 --- p.120 / Chapter 4.5.1 --- Purification of eEF2 from rat liver --- p.120 / Chapter 4.5.2 --- Physical interaction of P2 and eEF2 by surface plasmon resonance (SPR) --- p.126 / Chapter 4.5.3 --- Interaction between eEF2 and P2 mutants by in vitro binding assay --- p.128 / Chapter 4.6 --- Mapping the C-terminal region of P2 by MBP-fusion proteins --- p.130 / Chapter 4.6.1 --- Construction and purification of MBP-fusion proteins --- p.131 / Chapter 4.6.2 --- "Interaction among eEF2, TCS and MBP-fusion proteins by in vitro binding assay" --- p.133 / Chapter 4.7 --- Discussion --- p.137 / Chapter Chapter 5 --- Effect of C-17 peptide on TCS biological activity --- p.143 / Chapter 5.1 --- Introduction --- p.143 / Chapter 5.2 --- Ribosome-inactivating activity of TCS with C-17 peptide --- p.145 / Chapter 5.3 --- Discussion --- p.147 / Chapter Chapter 6 --- Conclusion and suggestions for future study --- p.149 / References --- p.152 / Appendix --- p.173

A selenocisteína (Sec, U) é o aminoácido que representa a principal forma biológica do elemento selênio e sua incorporação é um processo co-traducional em selenoproteínas como resposta ao códon UGA em fase e requer uma complexa maquinaria molecular. O repertório completo de genes envolvidos nessa via de síntese em procariotos é conhecido, porém algumas das interações moleculares ainda não foram totalmente esclarecidas. Este projeto visa à caracterização molecular nas interações entre o Fator de Elongação específico para incorporação de Sec (SelB) e Seril-tRNA sintetase (SerRS) com distintas construções do tRNASec de Escherichia coli afim de compreender a sua especificidade, seletividade e ordem de eventos. Para isso, medidas de Espectroscopia de Anisotropia de Fluorescência (FAS), Ultracentrifugação Analítica (AUC) e Calorimetria de Varredura Diferencial (DSC) foram utilizadas para determinação das constantes de interação desses complexos proteína-tRNA. Além disto, experimentos de Espalhamento de Raios-X a baixo ângulo (SAXS) e Microscopia eletrônica de transmissão por contraste negativo (NS-EM) foram realizados para elucidação estrutural destes complexos. Os estudos propostos irão auxiliar no entendimento do mecanismo de incorporação e de especificidade do tRNA para este aminoácido em bactérias bem como nos demais domínios da vida além de possibilitar um aumento na compreensão de complexos do tipo proteína-tRNA bem como salientar a importância dos elementos estruturais do tRNA para sua especificidade no processo de síntese de novas proteínas. / Selenocysteine (Sec, U) is an amino acid that represents the main biological form of the selenium element and its incorporation is a co-translational process in selenoproteins in response to the in-phase UGA codon and requires complex molecular machinery. The complete repertoire of genes involved in this pathway of synthesis in prokaryotes is known, although some of the molecular interactions have not yet been fully elucidated. This project aims at the molecular characterization in the interactions between the specific elongation factor for the incorporation of Sec (SelB) and Seril-tRNA synthase (SerRS) with different constructions of tRNASec from Escherichia coli in order to their specificity, selectivity and order of events. For this, measurements using Fluorescence Anisotropy Spectroscopy (FAS), Analytical Ultracentrifugation (AUC) and Differential Scanning Calorimetry (DSC) were employed to determine the interaction constants of the protein-tRNA complexes. In addition, Small Angle X-Ray Scattering (SAXS) experiments and negative stain transmission electron microscopy (NS-EM) were performed for structural elucidation of these complexes. The proposed studies will help to understand the mechanism of tRNA incorporation and specificity for this amino acid in bacteria as well as other domains of life. In addition, it allows an increase in the understanding of protein-tRNA-like complexes as well as emphasizing the importance of structural elements of tRNA for its specificity in the process of synthesis of new proteins.

A leptospirose é uma zoonose causada por bactérias patogênicas do gênero Leptospira. A doença representa um grave problema de saúde pública nos países tropicais subdesenvolvidos. Mais de 500.000 casos graves de leptospirose são notificados a cada ano e a taxa de mortalidade excede 10% (World Health Organization, 1999). Os roedores são o principal reservatório urbano da doença, e eliminam leptospiras viáveis no meio ambiente ao longo de toda a vida. As bactérias entram no hospedeiro por abrasões na pele ou por membranas mucosas e rapidamente se espalham pelo organismo atingindo vários órgãos. A identificação de mecanismos de invasão e de evasão imune apresentados por leptospiras patogênicas é extremamente relevante e tem sido alvo de pesquisas recentes desenvolvidas por vários grupos. Nesse contexto, a caracterização funcional de proteínas de membrana externa de Leptospira, principais alvos de interação com moléculas do hospedeiro, é de grande importância. O Fator de Elongação Tu (EF-Tu), uma proteína bacteriana abundante envolvida na síntese protéica, pertence à categoria das proteínas conhecidas como \"moonlighting\". Tais moléculas possuem a capacidade de exercer mais de uma função e, normalmente, localizam-se em diferentes compartimentos da célula. Há relatos de que EF-Tu de agentes patogênicos possa atuar como um fator de virulência. No presente trabalho, demonstrou-se que EF-Tu de Leptospira está localizado na superfície da bactéria e possui funções adicionais, sendo receptor para moléculas presentes no plasma do hospedeiro. Tal proteína interage com vários componentes da matriz extracellular e também com plasminogênio, de maneira dosedependente. Resíduos de lisina são importantes para essa interação. Plasminogênio ligado a EF-Tu é convertido em sua forma ativa, plasmina, que, por sua vez, é capaz de clivar os substratos naturais C3b e fibrinogênio. EF-Tu de Leptospira também se liga a Fator H, principal regulador da via alternativa do sistema complemento, e este mantém sua atividade funcional ao agir como co-fator de Fator I na clivagem de C3b. O potencial imunoprotetor de EF-Tu em modelo animal foi avaliado, tendo em vista o alto grau de conservação da proteína em diferentes espécies de Leptospira. EF-Tu não conferiu proteção significativa e, portanto, não deve ser considerado como um candidato vacinal contra a leptospirose. Em suma, EF-Tu de Leptospira deve contribuir para o processo de invasão e evasão ao sistema imune inato do hospedeiro, inativando o sistema complemento. Tanto quanto é do nosso conhecimento, essa é a primeira descrição de uma proteína \"moonlighting\" em Leptospira. / Leptospirosis is a zoonosis caused by pathogenic bacteria from the genus Leptospira. The disease represents a serious public health problem in underdeveloped tropical countries. More than 500,000 cases of severe leptospirosis are reported each year, with mortality rates exceeding 10% (World Health Organization, 1999). Rodents are the main urban reservoir of the disease, shedding viable leptospires throughout their lives in the environment. Leptospires infect hosts through small abrasions in the skin or mucous membranes and they rapidly disseminate to target organs. The identification of invasion mechanisms and immune evasion strategies employed by pathogenic leptospires is of great relevance. In this context, functional characterization of leptospiral outer membrane proteins, which represent the main targets for interaction with host molecules, is extremely important. The elongation factor Tu (EF-Tu), an abundant bacterial protein involved in protein synthesis, has been shown to display moonlighting activities. Known to perform more than one function at different times or in different places, it is found in several subcellular locations in a single organism, and may serve as a virulence factor in a range of important human pathogens. In this work we demonstrate that Leptospira EF-Tu is surface-exposed and performs additional roles as a cell-surface receptor for host plasma proteins. It interacts with several extracellular matrix components and also binds plasminogen in a dose-dependent manner. Lysine residues are critical for this interaction. Bound plasminogen is converted to active plasmin, which, in turn, is able to cleave the natural substrates C3b and fibrinogen. Leptospira EF-Tu also acquires Factor H (FH), the main soluble regulator of the alternative pathway of the complement system. FH bound to immobilized EF-Tu displays cofactor activity, mediating C3b degradation by Factor I (FI). Given the wide distribution of EF-Tu among Leptospira species, its immunoprotective potential was evaluated in an animal model. EF-Tu was not able to afford significant immunoprotection, and might not be considered a vaccine candidate against leptospirosis. In conclusion, EF-Tu may contribute to leptospiral tissue invasion and complement inactivation. To our knowledge, this is the first description of a leptospiral protein exhibiting moonlighting activities.

Pseudomonas aeruginosa is an opportunistic human pathogen that is now the leading cause of morbidity and mortality in immunocompromised individuals. Those suffering with the genetic disease cystic fibrosis (CF) commonly encounter P. aeruginosa infections. P. aeruginosa infection can present itself as an acute infection, which is characterised by highly virulent, "free-swimming" bacteria, or as a chronic infection associated with the formation of surface-adhered bacterial communities known as biofilms. The labyrinth of interconnecting signalling networks has meant that the regulatory mechanisms behind biofilm formation and virulence are largely undefined. In this dissertation, a single nucleotide polymorphism was identified within the gene, fusA1, encoding elongation factor G (EF-G). The mutation introduced minor structural changes to the protein which were likely to have functional repercussions in its involvement in protein synthesis. Phenotypic analysis revealed that the mutation conferred changes in both resistance and sensitivity to various antibiotics, as well as changes in motility, exoenzyme production, quorum sensing, metabolism, synthesis of biofilm-associated proteins and exopolysaccharide production. Most notably was the up-regulation of a major virulence determinant, the type three secretion system, typically characteristic of cells comprising an acute infection. Proteomic and transcriptomic profiling of the mutant strain provided an insight into the genetic basis behind these phenotypes, identifying the up-regulation of multidrug efflux systems and modulations to the chemotactic systems. This study also found links between several biological processes that were modulated in the mutant strain, such as crosstalk between sulfur metabolism, iron uptake and the oxidative stress response. In summary, the work presented in this dissertation highlights the susceptibility of fusA1 to spontaneous mutation and identifies a novel role for EF-G in bacterial virulence and antibiotic sensitivity, both of which have worrying implications for infection within the CF lung.

A leptospirose é uma zoonose causada por bactérias patogênicas do gênero Leptospira. A doença representa um grave problema de saúde pública nos países tropicais subdesenvolvidos. Mais de 500.000 casos graves de leptospirose são notificados a cada ano e a taxa de mortalidade excede 10% (World Health Organization, 1999). Os roedores são o principal reservatório urbano da doença, e eliminam leptospiras viáveis no meio ambiente ao longo de toda a vida. As bactérias entram no hospedeiro por abrasões na pele ou por membranas mucosas e rapidamente se espalham pelo organismo atingindo vários órgãos. A identificação de mecanismos de invasão e de evasão imune apresentados por leptospiras patogênicas é extremamente relevante e tem sido alvo de pesquisas recentes desenvolvidas por vários grupos. Nesse contexto, a caracterização funcional de proteínas de membrana externa de Leptospira, principais alvos de interação com moléculas do hospedeiro, é de grande importância. O Fator de Elongação Tu (EF-Tu), uma proteína bacteriana abundante envolvida na síntese protéica, pertence à categoria das proteínas conhecidas como \"moonlighting\". Tais moléculas possuem a capacidade de exercer mais de uma função e, normalmente, localizam-se em diferentes compartimentos da célula. Há relatos de que EF-Tu de agentes patogênicos possa atuar como um fator de virulência. No presente trabalho, demonstrou-se que EF-Tu de Leptospira está localizado na superfície da bactéria e possui funções adicionais, sendo receptor para moléculas presentes no plasma do hospedeiro. Tal proteína interage com vários componentes da matriz extracellular e também com plasminogênio, de maneira dosedependente. Resíduos de lisina são importantes para essa interação. Plasminogênio ligado a EF-Tu é convertido em sua forma ativa, plasmina, que, por sua vez, é capaz de clivar os substratos naturais C3b e fibrinogênio. EF-Tu de Leptospira também se liga a Fator H, principal regulador da via alternativa do sistema complemento, e este mantém sua atividade funcional ao agir como co-fator de Fator I na clivagem de C3b. O potencial imunoprotetor de EF-Tu em modelo animal foi avaliado, tendo em vista o alto grau de conservação da proteína em diferentes espécies de Leptospira. EF-Tu não conferiu proteção significativa e, portanto, não deve ser considerado como um candidato vacinal contra a leptospirose. Em suma, EF-Tu de Leptospira deve contribuir para o processo de invasão e evasão ao sistema imune inato do hospedeiro, inativando o sistema complemento. Tanto quanto é do nosso conhecimento, essa é a primeira descrição de uma proteína \"moonlighting\" em Leptospira. / Leptospirosis is a zoonosis caused by pathogenic bacteria from the genus Leptospira. The disease represents a serious public health problem in underdeveloped tropical countries. More than 500,000 cases of severe leptospirosis are reported each year, with mortality rates exceeding 10% (World Health Organization, 1999). Rodents are the main urban reservoir of the disease, shedding viable leptospires throughout their lives in the environment. Leptospires infect hosts through small abrasions in the skin or mucous membranes and they rapidly disseminate to target organs. The identification of invasion mechanisms and immune evasion strategies employed by pathogenic leptospires is of great relevance. In this context, functional characterization of leptospiral outer membrane proteins, which represent the main targets for interaction with host molecules, is extremely important. The elongation factor Tu (EF-Tu), an abundant bacterial protein involved in protein synthesis, has been shown to display moonlighting activities. Known to perform more than one function at different times or in different places, it is found in several subcellular locations in a single organism, and may serve as a virulence factor in a range of important human pathogens. In this work we demonstrate that Leptospira EF-Tu is surface-exposed and performs additional roles as a cell-surface receptor for host plasma proteins. It interacts with several extracellular matrix components and also binds plasminogen in a dose-dependent manner. Lysine residues are critical for this interaction. Bound plasminogen is converted to active plasmin, which, in turn, is able to cleave the natural substrates C3b and fibrinogen. Leptospira EF-Tu also acquires Factor H (FH), the main soluble regulator of the alternative pathway of the complement system. FH bound to immobilized EF-Tu displays cofactor activity, mediating C3b degradation by Factor I (FI). Given the wide distribution of EF-Tu among Leptospira species, its immunoprotective potential was evaluated in an animal model. EF-Tu was not able to afford significant immunoprotection, and might not be considered a vaccine candidate against leptospirosis. In conclusion, EF-Tu may contribute to leptospiral tissue invasion and complement inactivation. To our knowledge, this is the first description of a leptospiral protein exhibiting moonlighting activities.

Le but de cette thèse est l'étude expérimentale et théorique de l'élongation de M.R.E. (Magneto Rheological Elastomer) placé dans un champ magnétique homogène. Ces matériaux sont constitués de particules ferromagnétiques distribuées au sein d'une matrice élastique. La combinaison d'une matrice de silicone de faible module de Young (E0=0,14 MPa) combinée à la forte aimantation des particules de fer (µ0Msat=2,14 T) permet d'atteindre des déformations de plusieurs pourcents, pour un champ appliqué µ0H0=1,2 T. Le calcul des forces dipolaires entre les particules, distribuées aléatoirement dans un volume de forme cylindrique, couplé à un calcul de déformation (utilisant un logiciel F.E.M.) est en accord avec la mesure de magnetostriction. Un échantillon aimanté acquiert une énergie magnétique dite « démagnétisante » liée à sa forme : un échantillon « plat » aura une énergie démagnétisante plus importante qu'un échantillon « long ». L'aimantation d'un composite a été étudié dans cette thèse via 2 paramètres : l'aimantation à saturation et le coefficient de champ démagnétisant effectif. La mesure de déformation faite sur des échantillons de différentes formes montre l'effet de cette énergie démagnétisante : l'échantillon le plus plat (de facteur de forme c/a=0.3) se déforme ainsi jusqu'à près de 10 %. Un modèle basé sur la compétition entre l'énergie démagnétisante et l'énergie élastique, pendant la déformation, donne des valeurs de déformation prenant en compte cet effet de forme. Ce modèle prend en compte aussi l'effet de la fraction volumique sur la déformation du composite. Une concentration optimale de 27 % a été mesurée et prédite. La magnetostriction de composites avec des particules magnétiques dures a aussi été mesurée en fonction du champ. L'effet de l'hystérésis de ces particules génère un "effet mémoire" à la courbe de magnetostriction. Enfin, le comportement thermique de la magnetostriction de ces composites a été mesuré. La constante élastique de la matrice et l'aimantation des particules sont des fonctions de la température. Le rôle de ces paramètres permet de concevoir des matériaux avec différentes propriétés thermiques.

The deformation mechanism of an ultrafine grained (UFG) Al-Zn alloy has been studied. In this work, Al-15wt%Zn alloy was processed by equal channel angular extrusion (ECAE) route A at 100oC to achieve UFG structure. The deformation mechanism was studied by performing tensile test with various strain rates. Scanning electron microscopy and transmission electron microscopy were used to investigate the microstructure evolution in Al-15wt%Zn alloy with increasing ECAE passes. The observation indicated that the super saturated Al-Zn alloy would decompose and precipitate Zn particles during ECAE process. Increasing ECAE passes, the aluminum grain size was reduced, but the size of Zn particles was increased. However, the net effect of increasing ECAE passes is softening of this Al-Zn alloy. The tensile properties of the UFG Al-Zn alloy can be summarized as follows. (1)The UFG Al-Zn alloy possesses higher tensile strength and elongation as compared to commercial purity Al (AA1050). (2)The strain rate sensitivity of the UFG Al-Zn alloy increases significantly with increasing number of ECAE pass, which might be related to the refined aluminum grain size. After processed by 4-16 ECAE passes, the activation volume of the UFG Al-Zn alloy falls in the range of 25 b3~40 b3, which remains nearly constant value with increasing tensile strain. It is suggested that the controlling mechanism responsible for the tensile deformation of the UFG Al-Zn alloy might be related to a grain boundary mediated mechanism. (3)With increasing ECAE passes, the total tensile elongation of the UFG Al-Zn alloy increases but the uniform elongation show little change. This indicates that the increase in total elongation is mainly due to the contribution from an enhanced post-uniform elongation (PUE). It is suggested that the enhanced PUE might be related to the increase in strain rate sensitivity, which is resulted from the refinement of grain size. More detailed studies are needed to understand the deformation mechanism.