The inability of the myocardium to regenerate after injury plus the inadequate number of available hearts for transplantation have drawn attention to the creation of functional tissue constructs for implantation within the injured heart. In addition, there is an increasing interest in developing in vitro models to study heart physiology and pathology as well as to evaluate drug efficacy. Formation of these in vitro models and tissue constructs requires highly specific conditions to mimic the normal environment of cells in the body. Firstly, in this study, plasma lithography patterning of elastomeric substrates is exploited for creating microtissues composed of neonatal cardiomyocytes, and investigating their development in different mechanical microenvironments. Immunofluorescence microscopy and force spectroscopy show that the size and shape of the cardiomyocyte clusters, as well as the sarcomere length, fiber alignment, and beating amplitude and frequency of the cardiomyocytes, are regulated by microenvironmental cues. Computational analysis reveals that the mechanical stress at the cluster-substrate interface strongly correlates with the aforementioned characteristics of the cardiomyocytes. Taken together, our results underscore a collective mechanoadaptation scheme in cardiac development. Secondly, a silicone substrate with tunable elasticity is characterized for biological studies. Uniaxial tensile testing and microindentation show that these substrates could cover the biological range of stiffness for normal and pathological conditions. Spectrophotometry demonstrates that the transmittance of these substrates is comparable to those of glass and Sylgard 184. Atomic force microscopy shows that the surface roughness of samples is lower than that of widely-used Sylgard 184. Contact angle measurements before and after exposure to air plasma indicate that these samples are compatible with plasma lithography patterning. Thirdly, a new technique for cell patterning is developed which utilizes selective plasma lithography to modify protein adhesion on the substrate. This approach is based on controlling the conformation of Pluronic F-127 layer adsorbed on the surface by modifying surface wettability. Contact angle measurements show that both PDMS and plastic petri dish are compatible with this technique. X-ray photoelectron spectroscopy and atomic force microscopy confirm the adsorption of PF-127 layers with controlled conformation. Fluorescent and bright-field microscopy demonstrate selective adhesion of proteins and attachment of cells merely on plasma-treated areas. Finally, micropillar arrays are employed to determine the effects of two proteins associated with regulation of thin filament length, i.e. Lmod2 and Tmod1, on contractile force generation at the cellular level. Our results demonstrate that the contractile force of single isolated Lmod2-KO cardiomyocytes decreases compared to the wildtype control. Transduction of Lmod2 in the knockout cardiomyocytes restores their contractile force to the level of their WT counterparts, verifying that the observed contractile dysfunction is specific to the loss of Lmod2. Our data demonstrate that overexpression of Tmod1 in cardiomyocytes decreases their contractile force compared to the WT cells and confirm the effects of Lmod2 knockout on contractile force generation.

L'ablation par radiofréquence constitue un des traitements des tachycardies ventriculaires, en association avec les drogues anti-arythmiques et l’implantation d'un défibrillateur. L’objectif principal de cette thèse est de mieux comprendre le substrat arythmogène non seulement à l’aide d'imagerie cardiaque (IRM et scanner) de haute résolution et de cartographie de haute densité, en utilisant des cathéters multipolaires. Cela nous permettra d'analyser la relation structure-fonction. Nous avons étudié cette relation sur différents types de substrats (ICM, NICM, DAVD, et myocardites). Nous avons ainsi prouvé la supériorité de la cartographie de haute densité obtenue à partir de cathéters multipolaires, comparativement aux données recueillies par l’imagerie, dans l’identification de la cicatrice arythmogène et la détection des LAVA. La deuxième partie de cette thèse concerne l’étude du substrat arythmogène épicardique. Nous avons ainsi décrit la technique de cartographie par voie percutanée antérieure, puis démontré l'efficacité des procédures uniquement avec abord épicardique. La segmentation du nerf phrénique et des artères coronaires ont permis de diminuer le taux de complications théoriquement liés à cet abord. Nous avons poursuivi ce travail avec l’analyse des sites d'intérêt de l'ablation des TV: les LAVA. Après une description de la stratégie d’élimination des LAVA, nous avons tenté de trouver des prédicteurs permettant de localiser les sites de LAVA, à partir des données d'imagerie. Quand l'imagerie montre une cicatrice intraseptale ou intramurale, les LAVA ne peuvent pas être enregistrés avec la cartographie et des alternative techniques d'ablation sont nécessaires comme une ablation bipolaire, l'alcoolisation intra coronaire et l'ablation avec l'aiguille irriguée. Le dernier chapitre est une revue sur le futur de l'imagerie, de la cartographie et de l’ablation des tachycardies ventriculaires. Une meilleure compréhension du substrat arythmogène pourrait améliorer l'efficacité et la sécurité des ablations de tachycardie ventriculaire. / Radiofrequency (RF) catheter ablation is an effective treatment strategy for scar-related ventricular tachycardia (VT), resistant to anti-arrhythmic drugs and intracardiac defibrillator (ICD) placement. The goal of this thesis was to better understand and characterize the arrhythmogenic VT substrate in different cardiomyopathic processes: ischaemic cardiomyopathy (ICM), non-ischaemic cardiomyopathy (NICM), arrhythmogenic right ventricular cardiomyopathy (ARVC) and myocarditis. For this purpose, we combined high resolution imaging including different modalities and high resolution electrical mapping to better understand the structure-function relationship. We focused on multiple different aspects of VT ablation as outlined below. The first part of this thesis focuses on the role of multipolar mapping catheters and imaging to analyze their structural and functional relationship. We demonstrated superiority of high density mapping with multipolar mapping on conventional mapping in detection of scar, channels, local abnormal ventricular activity (LAVA) and sensitivity for near field signals. The second part of this thesis focuses on ablation of epicardial VT substrate. We demonstrated the efficacy and safety of epicardial only procedures in a highly selected population. We used imaging to have access to the exact anatomy of the heart, to image the substrate but also to increase the safety of ablation procedures by imaging the phrenic nerve and the coronary artery system. The third part of this thesis focuses on analysis of the mapping and ablation of potential targets for scar-related VT ablation. Within this context, we identified predictors of interesting ablation (LAVA) sites based on preprocedural imaging. We also analyzed the role of alternative strategies such as bipolar ablation, ethanol ablation and irrigated needle ablation to ablate intramural and intraseptal substrate, 18 often resistant ablation targets. Overall, we demonstrate that novel imaging, mapping and ablation techniques potentially improve the outcome of VT ablation.

The objective of this study was to evaluate the effect of nitrogen concentration on the production and quality of strawberry bare root transplants and runner tips in a soilless growing system using sand as substrate. Stock plants were planted on September 13th, 2007. Transplants were harvested on February 18th, 2008. Treatments were nitrogen concentrations in the nutrient solution of 8, 11, 14 and 17 mmol L-1. The experiment was a completely randomized design with three replications of 3.6m2 plots. Number and crown diameter of bare root transplants and number of runner tips were evaluated at harvesting. Number of bare root transplants and runner tips was not affected by N concentration in the nutrient solution. An average of 339 bare root transplants and 836 runner tips were harvest per stock plant. Crown diameter of bare root transplants decreased linearly with the increase in N concentration of the nutrient solution. It was concluded that in this soilless cropping system the increase in N concentration in the nutrient solution does not affect the number of strawberry bare root transplants and runner tips, but the crown diameter of bare root transplants is reduced. The N concentration of 8 mmol L-1 may be used for crop propagation in this growing system. / O objetivo do trabalho foi avaliar o efeito da concentração de nitrogênio na produção e na qualidade de mudas de raízes nuas e pontas de estolões de morangueiro no cultivo sem solo empregando areia como substrato. O plantio das matrizes foi realizado em 13 de setembro de 2007 e a colheita das mudas em 18 de fevereiro de 2008. Os tratamentos foram quatro concentrações de N na solução nutritiva de 8, 11, 14 e 17 mmol L-1. O delineamento utilizado foi o inteiramente casualizado, com três repetições de 3,6m2. No momento da colheita foi determinado o número e o diâmetro da coroa das mudas de raízes nuas e o número de pontas de estolões. As concentrações de N não afetaram o número de mudas e de pontas de estolões, cujas médias foram de 339 e 836, respectivamente. O diâmetro da coroa das mudas de raízes nuas diminuiu linearmente com o aumento da concentração de N. Concluiu-se que o aumento da concentração de N na solução nutritiva nesse sistema de cultivo sem solo não afeta o número de mudas de raízes nuas e nem de pontas de estolões, mas reduz o diâmetro da coroa das mudas de raízes nuas de morangueiro. A concentração de 8 mmol.L-1 de N pode ser empregada para fins de produção de mudas nesse sistema.

The goal of this work was to determine the N requirements of the muskmelon crop and adjusting a method for diagnosis of the plant nutritional status, for estimations of fertilization rates to be used during growth and development of this crop. The experiment was carried out at Departamento de Fitotecnia - UFSM, inside a 500m2 screenhouse. Muskmelon plantlets, hybrid Magelan, were planted at September 30th, 2004, in 4.5dm3 plastic bags containing a commercial substrate. Plant density was 3.3 plants m-2. Water and nutrients were supplied by fertigation. Treatments were five nutrient solutions with N concentrations (NO3 - /NH4 + ) of 8 (T1), 11 (T2), 14 (T3), 17 (T4) and 20mmol.L-1 (T5). The other nutrients were supplied at standard concentrations of 0.9 H2PO4 - ; 2.25 SO4 -- ; 10.0 Ca++; 6.0 K+ and 5.0mmol.L-1 Mg++, with micronutrients. Four plants were weekly harvested from 33 to 99 days after planting (DAP) to determine dry mass (DM), number of fruits (NF), leaf area index (LAI) and N content in tissues. Ripe fruits were harvested at the peduncle abscission stage and fresh weight, flesh firmness, fraction of flesh, skin, mucilage and seeds, titratable acidity and soluble solids were determined. The N content in leaf, stem, petiole and fruit tissues was determined by the Kjeldahl method. The vegetative and total dry mass, leaf area, number and fruit yield increased linearly by effect of treatments. For fruit quality variables, only the titratable acidity decreased by increasing N concentrations. The N dilution was observed during growth of tissues upon all treatments and data fitted the potential model %N = aMS-b described in the literature. The N critical dilution curve was adjusted, with values of 5.16 and 0.63 for model coefficients a and b, respectively. It was concluded that N affects the yield of the crop and can be supplied at rates estimated from the critical dilution curve until fruit setting was attained. After this stage, N fertilization rates can be adjusted in order to avoid any restrictions in the leaf area growth. / Os objetivos deste trabalho foram determinar as necessidades de N pela cultura do melão e ajustar um método de diagnóstico das doses de adubação no decorrer do ciclo de crescimento e desenvolvimento. O experimento foi conduzido no Departamento de Fitotecnia da UFSM, no interior de um abrigo de 500m2, coberto com polietileno aditivado de 150μm de espessura. Mudas de melão do híbrido Magelan foram plantadas em 30/09/2004 em sacolas de polietileno com 4,5dm3 de substrato comercial, na densidade de 3,3 plantas.m-2 as quais foram fertirrigadas com solução nutritiva completa. Os tratamentos consistiram por cinco concentrações de nitrogênio (NO3 -/NH4) de 8 (T1), 11 (T2), 14 (T3), 17 (T4) e 20mmol. L-1 (T5). Os demais nutrientes foram fornecidos nas mesmas concentrações para os cinco tratamentos, sendo de 0,9 de H2PO4 - ; 2,25 de SO4 -- ; 10,0 de Ca++; 6,0 de K+ e 5,0mmol L-1de Mg++, complementadas por micronutrientes. Para determinação da massa seca (MS), número de frutos (NF), índice de área foliar (IAF) e o teor de Nitrogênio nas folhas, haste, pecíolos e frutos, foram coletadas semanalmente quatro plantas de cada tratamento no período entre 33 e 99 dias após o plantio (DAP). A massa seca de cada órgão foi moída em moinho tipo Willey e a concentração de N determinada pelo método Kjeldah. A colheita foi feita diariamente quando os frutos evidenciaram a zona de abscisão completa em torno do pedúnculo. As características avaliadas foram o peso de fruto, firmeza da polpa, as frações da polpa, casca, mucilagem e sementes, acidez titulável (AT) e o teor de sólidos solúveis totais (TSS). Os resultados foram submetidos à análise da variância e os dados das variáveis que apresentaram diferenças significativas entre os tratamentos foram submetidos à análise de regressão. Foi observada resposta linear dos tratamentos na massa seca vegetativa e total, área foliar, número de frutos e produtividade. Quanto às características qualitativas dos frutos, somente a acidez titulável apresentou diferença significativa, decrescendo com o aumento da concentração de N. Foi observada a diluição da concentração de N na massa seca em todos os tratamentos e os dados ajustaram-se ao modelo potencial %N = aMS-b descrito na literatura. A curva crítica de diluição do N foi ajustada, com coeficientes a e b iguais a 5,16 e 0,63, respectivamente. Concluiu-se que o N interfere na produtividade e deve ser fornecido em doses não superiores à curva crítica de diluição até a que a fixação dos frutos tenha sido atingida. Após essa fase doses devem ser ajustadas de forma a evitar restrições no crescimento da área foliar e dos frutos.

Abstract In the cells, the last step of the beta-oxidation cycle, aiming at the degradation of fatty acids, is catalyzed by the enzyme named thiolase. It shortens the acyl chain of the acyl-CoA by two carbons. The reaction is reversible, it can proceed for both directions. Thiolases are divided into two categories, synthetic and degradative ones. These two classes of thiolases differ not only by their biological function, but also by their substrate specificity. Degradative thiolases accept substrates with various lengths but synthetic thiolases only accept short chain-acyl-CoAs as a substrate. In humans, at least six isozymes of thiolases are found. The mitochondrial biosynthetic thiolase, T2, differs from other thiolases by getting activated by potassium. In addition, it accepts branched acyl-CoA, namely 2-methyl-acetoacetyl-CoA, as a substrate. This molecule is an important reaction intermediate in the degradation of the amino acid isoleucine. Many human patients have been diagnosed to have a mutation in the gene of T2, and they are treated with a special diet. The results of this theses show that potassium ion rigidifies the groups of the T2 protein involved in the substrate binding. The presence of potassium increases the reaction rate and it also raises the affinity towards some of the substrates. The enzyme mechanistic studies with bacterial thiolase revealed that the oxyanion hole 1, formed by a water molecule and histidine side chain, is important for the synthetic reaction, not so much for the degradative direction. Binding studies showed that both the terminal sulfur of the substrate and the sulfur of the catalytic cysteine are important for the right positioning of the substrate. The electrostatics of the active site also have a significant role in the catalysis. These studies give a good basis for future studies aiming at drug development against this enzyme in pathogenic species.

Mining and construction industry is a major environmental disturbance creating habitats with extremely coarse material: gravel, cobbles and boulders. Simultaneously, there is an increasing interest in using spontaneous succession during restoration processes on those areas. This leads to a question if vegetation can establish on such coarse substrates. This project assessed the vegetation establishment on substrates with different grain size as well as the hydrological limitations of those substrates. The aim of this study was to find substrate moisture characteristics supporting vegetation establishment. Four herb species (Koeleria macrantha, Epilobium angustifolium, Artemisia campestris var. campestris and Poa nemoralis) typically occurring on xerothermic basalt rocks were sowed on basalt construction aggregates (0-4, 2-4, 4-8, 8-16, 11-22 and 32-63 mm). There were also made mixtures of coarse aggregates (4-8, 8-16 and 11-22 mm) as a base with 10% or 50% additive of fine aggregates (0-4 mm). The final number of individuals, aboveground and belowground dry biomass were measured and subsequently specific leaf area (SLA) was calculated. They were also measured substrate moisture characteristics: field capacity (PVK) and wilting point (BV) from which available water content (ODV) was obtained as...

La décomposition des résidus végétaux joue un rôle essentiel dans le cycle biogéochimique des éléments nutritifs et influence le fonctionnement des écosystèmes. La composition biochimique intrinsèque des résidus végétaux est un facteur clé qui influe sur les processus de décomposition dans le sol tandis que la majorité des réactions biochimiques dans le sol, liées à la biodégradation des résidus végétaux, sont catalysées par des enzymes extracellulaires produites par les microorganismes. L'objectif global de cette étude était d'acquérir des connaissances fondamentales concernant l'impact de la qualité des résidus sur les fonctions microbiennes du sol et les modes d'intervention des enzymes du sol interviennent dans la décomposition des résidus végétaux. Cet objectif a été atteint en trois parties visant à : 1) déterminer le rôle des communautés initiales des résidus i.e. les microorganismes et leurs enzymes provenant des compartiments épiphytes et endophytes, et l'effet de la qualité des résidus végétaux sur les cinétiques des enzymes extracellulaires au cours du processus de décomposition dans le sol 2) étudier l'effet des fonctions microbiennes du sol (biomasse microbienne et enzymes extracellulaires) liées à la minéralisation sur la décomposition ultérieure de résidus introduits dans le même sol 3) explorer les interactions entre la disponibilité en azote et la décomposition des composés phénoliques par l'action des activités oxydo-réductases, et développer une méthode pour mesurer ces activités dans des sols contrastés en utilisant un seul substrat. L'approche générale de cette étude a été de sélectionner les résidus végétaux de qualité chimique variable pour obtenir des cinétiques contrastées de minéralisation du C. Le maïs (Zea mays L.) a été choisi comme plante modèle en raison de variations chimiques et structurales (Mexxal, F2, F2bm1, F292bm3) des parties aériennes (feuilles, entre-nœuds) et souterraines (racines). Des tiges de lin marqué au 13C ont été utilisées pour quantifier avec précision la minéralisation du carbone dans les différents réservoirs de carbone. Afin d'évaluer les relations entre la qualité des résidus végétaux et les fonctions biologiques associées au sol, des expériences en microcosmes contrôlés ont été réalisées en utilisant des sols agricoles et forestiers. La minéralisation du carbone, les caractéristiques chimiques des résidus (teneurs en C et N, les sucres totaux et lignine), la biomasse microbienne et les activités enzymatiques (L-leucine aminopeptidase (LAP), cellobiohydrolase (CBH-1), xylanase, cellulase et la laccase) ont été déterminées à différents stades de décomposition. Les résultats de la première étude ont indiqué que les activités de micro-organismes épiphytes et endogènes étaient du même ordre de grandeur dans le cas des racines, tandis que les activités des enzymes spécifiques (cellulase, xylanase et laccase) étaient fortement corrélées à la dégradation de leurs substrats cibles (glucanes, xylanes et lignine, respectivement). Dans la seconde étude, l'addition répétée de résidus a eu peu d'effet sur la biomasse microbienne et la dynamique enzymatique, sauf la LAP et la laccase. Ces résultats suggèrent que la qualité des résidus végétaux est le principal facteur déterminant les modes d'action de la biomasse microbienne et de leurs enzymes extracellulaires durant le processus de décomposition dans le sol. Les résultats de la dernière étude ont démontré que l'addition d'azote réprimait la minéralisation du carbone des résidus les moins lignifiés (F2, F2bm1), mais n'a pas affecté celle du résidu plus lignifié (F292bm3) au cours de la décomposition à long terme. L'ABTS est apparu comme un meilleur substrat que le L-DOPA, le pyrogallol et le TMB pour estimer les activités phénoloxydase et peroxydase.Mots clés: décomposition, biomasse microbienne, enzymes extracellulaires, qualité des résidus, maïs. / Plant residue decomposition plays a pivotal role in the biogeochemical cycling of nutrients and influences ecosystem functioning. The intrinsic biochemical composition of plant residues is a key factor influencing decomposition processes in soil while the majority of biochemical reactions in soil, related to the biodegradation of plant residues, are catalyzed by extracellular enzymes produced by microorganisms. The overall goal of this research study was to gain fundamental knowledge regarding the impact of residue quality on soil microbial functions and the principles by which soil enzymes mediate plant residue decomposition. This goal was achieved in three parts: 1) to determine the role of the initial residue community i.e. microorganisms and enzymes from the epiphytic and endophytic compartments and effect of plant residue quality on the extracellular enzyme kinetics during the decomposition process in soil 2) to investigate the effect of soil microbial functions (microbial biomass and extracellular enzymes) on the subsequent residue decomposition in the same soil 3) to explore the interactions between nitrogen availability and the decomposition of phenolic compounds through the action of oxydo-reductase enzymes activities and to develop a method to measure these activities in contrasted soils using a single substrate. The general approach of this study was to select plant residues with variations in their chemical quality to obtain contrasted C mineralization kinetics. Maize (Zea mays L.) was selected as a model plant because of variations in chemical and structural characteristics (Mexxal, F2, F2bm1, F292bm3) of aerial (leaves, internodes) and underground parts (roots). 13C-labeled flax stems were used to quantify accurately carbon mineralization in different carbon pools. To assess the relationships between plant residue quality and associated soil biological functions, controlled microcosm experiments were performed using agricultural and forest soils. Carbon mineralization and chemical characteristics (C and N contents, total sugars and lignin contents) of the plant residue, microbial biomass and enzyme activities (L-leucine aminopeptidase (LAP), cellobiohydrolase (CBH-1), xylanase, cellulase and laccase) were determined at different stages of decomposition. The results of first study indicated that activities of epiphytic and endogenic microorganisms were of the same order of magnitude in case of roots while the activities of specific enzymes (cellulase, xylanase and laccase) were highly correlated to the degradation of their target substrates (glucans, xylans and lignin, respectively). In the second study, little effect of repeated residue addition was observed on microbial biomass and enzyme dynamics except LAP and laccase. These results suggest that plant residue quality is the main factor which determines the fate/patterns of microbial biomass and their extracellular enzymes during decomposition process in soil. The results of last study demonstrated that nitrogen addition repressed the carbon mineralization of less lignified residues (F2, F2bm1) but did not affect more lignified residue (F292bm3) in long term decomposition. For estimation of phenol oxidase and peroxidase activities, ABTS appeared as a better substrate than L-DOPA, pyrogallol and TMB.Key words: decomposition, microbial biomass, extracellular enzymes, residue quality, maize.

Although the regulation of proteins functions by allosteric interactions has been identified in many subcellular processes, long-range conformational changes in proteins are also known to be induced by molecular switches. A molecular switch based on the cis-trans isomerization of a peptidyl-prolyl bond is capable of inducing a conformational change directly to the protein backbone, which is then propagated throughout the system. However, these switches are elusive and difficult to identify due to their intrinsic dynamics in the biomolecules where they are found. Herein, we explore the conformational dynamics and free energy landscape of the SH2 domain of Interleukin-2-inducible T-Cell Kinase (ITK) to fully understand the conformational coupling between the distal cis-trans molecular switch, and its phosphotyrosine binding pocket. Using multiple microsecond-long all-atom molecular dynamics simulations in explicit water for over a total of 60 μs, we show that the cis-trans isomerization of the Asn286-Pro287 peptidyl-prolyl bond is directly correlated to the dynamics of the phosphotyrosine binding pocket, in agreement with previous NMR studies. While the cis state is localized to a single free energy basin and less dynamic, the trans state samples two distinct conformations of its binding pocket – one that recognizes the phosphotyrosine motif, and another that is similar the cis state. These results provide an atomic-level description of a less-well understood allosteric regulation by a peptidyl-prolyl cis-trans molecular switch that could aid in the understanding of normal and aberrant sub-cellular process and the identification of these elusive molecular switches in other proteins.

No description available.

The human protein kinase superfamily consists of over 500 members that individually control specific aspects of cell behavior and collectively control the complete range of cellular processes. That such a large group of proteins is able to uniquely diversify and establish individual identities while retaining common enzymatic function and significant sequence/structural conservation is remarkable. The means by which this is achieved is poorly understood, and we have begun to examine the issue by performing a comparative analysis of the catalytic domain of protein kinases. A novel approach for protein structural alignment has revealed a high degree of similarity found across the kinase superfamily, with variability confined largely to a single region thought to be involved in substrate binding. The similarity detected is not limited to amino acids, but includes a group of conserved water molecules that play important structural roles in stabilizing critical residues and the fold of the kinase domain. The development of a novel technique for identifying kinase substrates on a large scale directly from cell lysate has revealed that substrate specificity is not what discriminates the closely related p38α and β mitogen-activated protein kinases. Instead cellular localization appears to be their distinguishing characteristic, at least during myoblast differentiation. Together these results highlight the extent of conservation, as well as the minimal variability, that is found in the catalytic domain of all protein kinase superfamily members, and that while distantly related kinases may be distinguished by substrate specificity, closely related kinases are likely to be distinguished by other factors. Although these results focus on representative members of the kinase superfamily, they give insight as to how all protein kinases likely diversified and established unique non-redundant identities. In addition, the novel techniques developed and presented here for structural alignment and substrate discovery offer new tools for studying molecular biology and cell signaling.