Conselho Nacional de Desenvolvimento Científico e Tecnológico / The objective of this research was to evaluate tree pasture-based systems (PS) with elephant grass (EG) + spontaneous growing species (SGS), ryegrass (RG), for PS1; EG + SGS + forage peanut, for PS2; and EG + SGS + RG + red clover, for PS3. EG was planted 4 m spaced rows. In the cool-season, ryegrass was sowed between rows of EG; red clover was sowed and the forage peanut was preserved in respectively PS. Experimental design was completely randomized with tree treatments (SF), two replicates (paddocks) in completely split-plot time (grazing cycles). Holstein cows receiving 5.5 kg-daily and a complementary concentrate feed were used in the evaluation. The herbage mass parameters, botanical composition, forage production and stocking rate were evaluated. Samples were collected by the hand-plucking method to analyze the organic matter (OM), crude protein (CP), neutral detergent fiber (NDF) and organic matter in situ digestibility (OMISD) of pasture; phenols and tannins on legumes. Nine grazing cycle were performed during the experimental period (341 days). The mean values of forage production, stocking rate, crude protein and digestibility were 17.14; 16.80; and 19.47 t/ha; 3.28; 3.34; and 3.60 UA/ha; 13.86; 15.39; and 14.78%; 78.33; 79.23; and 79.94% on the respective PS. SGS increased significantly (P≤0,05) relation to the PS without forage legume. Similar results were observed for forage production OM an NDF in comparison to PS. Nutritive value was similar between forage legumes. Differences (P≤0,05) on condensed tannin tenors were observed between forage legumes. Considering the forage production, stocking rate and nutritive value, the PS mixed to forage legumes showed better performance. / O objetivo desta pesquisa foi avaliar três sistemas forrageiros (SF) com capim elefante (CE), espécies de crescimento espontâneo (ECE), azevém anual (AZ), como SF1; CE + ECE + AZ + amendoim forrageiro (AF), como SF2; e CE + ECE + AZ + trevo vermelho (TV), como SF3. O capim elefante foi estabelecido em linhas afastadas a cada 4 m. No período hibernal fez-se o estabelecimento do AZ entre as linhas do capim elefante; o trevo vermelho foi semeado e o amendoim forrageiro foi preservado, considerando os respectivos tratamentos. O delineamento experimental foi o inteiramente casualizado, com três tratamentos (SF), duas repetições (piquetes) em parcelas subdivididas no tempo (pastejos). Para avaliação foram usadas vacas da raça Holandesa que receberam complementação alimentar com concentrado à razão de 5,5 kg/dia. Foram avaliados parâmetros da massa de forragem pré-pastejo, a composição botânica, a produção de forragem e a lotação. Para analisar a matéria orgânica (MO), a fibra em detergente neutro (FDN), proteína bruta (PB) e digestibilidade in situ da MO (DISMO) da pastagem e os teores de fenóis e taninos das leguminosas, foram coletadas amostras de pastejo simulado. Durante o período experimental (341 dias) foram efetuados nove ciclos de pastejo. Os valores médios de produção de forragem, lotação, PB e de DISMO foram 17,14; 16,80; e 19,47 t/ha; 3,28; 3,34; e 3,60 UA/ha; 13,86; 15,39; e 14,78%; 78,33; 79,23; e 79,94% nos respectivos SF. Houve aumento significativo (P≤0,05) para espécies de crescimento espontâneo na pastagem sem leguminosa. Resultados similares foram observados entre SF para MO e FDN. O valor nutritivo foi similar entre as leguminosas forrageiras. Foram observadas diferenças (P≤0,05) entre as leguminosas para os teores de taninos condensados. Considerando a massa de forragem, a lotação e o valor nutritivo, as pastagens consorciadas com leguminosas forrageiras apresentaram melhor desempenho.

Heteropolymeric O antigen (O-Ag)-capped lipopolysaccharide is the principal constituent of the Gram-negative bacterial cell surface. It is assembled via the integral inner membrane (IM) Wzx/Wzy-dependent pathway. In Pseudomonas aeruginosa, Wzx translocates lipid-linked anionic O-Ag subunits from the cytoplasmic to the periplasmic leaflets of the IM, where Wzy polymerizes the subunits to lengths regulated by Wzz1/2. The Wzx and Wzy IM topologies were mapped using random C-terminal-truncation fusions to PhoALacZα, which displays PhoA/LacZ activity dependent upon its subcellular localization. Twelve transmembrane segments (TMS) containing charged residues were identified for Wzx. Fourteen TMS, two sizeable cytoplasmic loops (CL), and two large periplasmic loops (PL3 and PL5 of comparable size) were characterized for Wzy. Despite Wzy PL3–PL5 sequence homology, these loops were distinguished by respective cationic and anionic charge properties. Site-directed mutagenesis identified functionally-essential Arg residues in both loops. These results led to the proposition of a “catch-and-release” mechanism for Wzy function. The abovementioned Arg residues and intra-Wzy PL3–PL5 sequence homology were conserved among phylogenetically diverse Wzy homologues, indicating widespread potential for the proposed mechanism. Unexpectedly, Wzy CL6 mutations disrupted Wzz1-mediated regulation of shorter O-Ag chains, providing the first evidence for direct Wzy–Wzz interaction. Mutagenesis studies identified functionally-important charged and aromatic TMS residues localized to either the interior vestibule or TMS bundles in a 3D homology model constructed for Wzx. Substrate-binding or energy-coupling roles were proposed for these residues, respectively. The Wzx interior was found to be cationic, consistent with translocation of anionic O-Ag subunits. To test these hypotheses, Wzx was overexpressed, purified, and reconstituted in proteoliposomes loaded with I−. Common transport coupling ions were introduced to “open” the protein and allow detection of I− flux via reconstituted Wzx. Extraliposomal changes in H+ induced I− flux, while Na+ addition had no effect, suggesting H+-dependent Wzx gating. Putative energy-coupling residue mutants demonstrated defective H+-dependent halide flux. Wzx also mediated H+ uptake as detected through fluorescence shifts from proteoliposomes loaded with pH-sensitive dye. Consequently, Wzx was proposed to function via H+-coupled antiport. In summary, this research has contributed structural and functional knowledge leading to novel mechanistic understandings for O-Ag biosynthesis in bacteria. / Bookmarks within the document have been provided for ease of access to a particular section in the body of the thesis. Each entry in the Table of Contents, List of Tables, and List of Figures has been "linked" to its respective position and as such can be clicked for direct access to the entry. Similarly, each in-text Figure or Table reference has been "linked" to its respective figure/table for direct access to the entry. / 1.) Canadian Institutes of Health Research (CIHR) Frederick Banting and Charles Best Canada Graduate Scholarship doctoral award, 2.) CIHR Michael Smith Foreign Study Award, 3.) Cystic Fibrosis Canada (CFC) doctoral studentship, 4.) University of Guelph Dean's Tri-Council Scholarship, 5.) Ontario Graduate Scholarship in Science and Technology, 6.) Operating grants to Dr. Joseph S. Lam from CIHR (MOP-14687) and CFC

Ce travail de thèse se focalise sur le couplage du microscope à force atomique haute–vitesse (HS-AFM) et de la spectroscopie Raman exaltée de surface (SERS) pour la détection des biomolécules. Nous avons élaboré un protocole de fabrication pour produire les substrats “SERS-actifs”. L’efficacité des substrats de nanoparticules cristalline d’or, d’argent ou bimétallique argent–or a été évaluée. Nous avons étudié l’impact des propriétés optiques et morphologiques des substrats sur l’intensité Raman en analysant des échantillons tests tels que la bipyridine éthylène et le bleu de méthylène. Nous nous sommes interessés à trois problematiques biologiques distinctes par analyses HS-AFM et SERS. Dans un premier cas, nous avons détecté la signature chimique de protéine cytochrome b5. Ce travail a été suivi par des études sur le changement de conformation de la protéine de choc thermique leuconostoc oenos Lo 18 en fonction de la concentration et du pH. La dernière application consiste en l’analyse des interactions membrane – virus. Afin de réaliser les analyses simultanées Raman/AFM, nous avons adapté notre protocole de fabrication pour couvrir la surface des pointes AFM commerciales par des nanoparticules d’or cristallines. Les études de diffusion Raman exaltée par effet de pointe (TERS) ont été effectuées sur les échantillons de disulfure de molybdène pour évaluer la qualité des pointes TERS. Pour finir, nous présentons une nouvelle configuration de couplage HS-AFM et spectroscopie Raman. Nous discutons des modifications et des défis rencontrés. / This thesis focuses on the coupling of High–Speed Atomic Force Microscopy (HS-AFM) and Surface Enhanced Raman Spectroscopy (SERS) for biomolecule analysis. We have designed a fabrication protocol to manufacture “SERS-active” substrates. The efficacy of gold, silver and gold-silver bimetallic crystalline nanoparticle substrates were evaluated. We have investigated the impact of optical and morphological features of the substrates on Raman signal intensity by analyzing well-known samples such as bipyridine ethylene and methylene blue molecules. We took an interest in three distinct biological problematics with HS-AFM and SERS analyses. First, we have detected the chemical signature of cytochrome b5 protein. This study was followed by the investigation of conformational changes of small heat shock leuconostoc oenos Lo 18 protein in function of pH level and concentrations. The last application consists to the analyse a membrane and a virus interaction. In order to realize simultaneous Raman/AFM analysis, we have adapted our fabrication protocol to cover the surface of commercial AFM probes by crystalline gold nanoparticles. Tip – Enhanced Raman Spectroscopy (TERS) studies were performed on molybdenum disulfide to evaluate the quality of TERS probes. In the last part of this work, we have designed a new setup to combine Ando’s HS-AFM setup with Raman spectroscopy. We present the modifications that have been carried out and the challenges that we have encountered.