<p>Hedgehog related genes have been shown to play a major role in development in all deuterostomes. In C.elegans, such genes have been found where the similarity is restricted to the C-terminal domain. This work has focused on the hedgehog related C.elegans proteins called ground (grd), ground-like (grl), and wart (wrt) which appear to form a unique structural family.These proteins are cysteine rich and have conserved cysteine patterns which, together with thethought that they are secreted, are expected to be in disulfide form. Since the extracellular environment is very oxidizing and due to the conserved cysteine pattern, disulfide bonds are thought to play a big part in the folding and stabilization of these proteins. The stability of the protein and the formation of a disulfide bond are related through a thermodynamic cycle, which insures that the stabilization of the protein by the disulfide is reflected by the identical stabilization of the disulfide by the protein. Practically, there are numerous parameters that can be used to try to achieve the correct disulfide bonds and folding, when doing in vitro trials, some of which were used in this project. C.elegans proteins grd-5, grd-13, grl-24, wrt-3 and wrt-5 were studied in this project. All of the proteins were expressed and purified with success, with theexception of grl-24. All constructs formed inclusion bodies. Some refolding attempts were performed on grd-13 and wrt-3. The presence of a disulfide bond in refolded grd-13 was demonstrated using chemical fragmentation. In general, these attempts did not give correctly folded proteins but provide a foundation to continue experiments aimed at producing a native-like protein for structural and functional studies.</p>

Micro/nano-patterning of supported lipid bilayers (SLBs) has shown considerable potential for addressing fundamental biophysical questions about cell membrane behavior and the creation of a new generation of biosensors. Herein are presented several novel lithographic methods for the size-controlled patterning of SLBs from the microscale to the nanoscale. Using these methods, chemically distinct types of phospholipid bilayers and/or Escherichia Coli (E. Coli) membranes can be spatially addressed on a single microchip. These arrays can, in turn, be employed in the studies of multivalent ligand-receptor interactions, enzyme kinetics, SLBs size limitation, and membrane-associated species separation. The investigations performed in the Laboratory for Biological Surface Science include the following projects. Chapters II and III describe the creation of lab-on-a-chip based platforms by patterning SLBs in microfluidic devices, which were employed in high throughput binding assays for multivalent ligand-receptor interactions between cholera toxin B subunits (CTB) and ganglioside GM1. The studies on the effect of ligand density for multivalent CTB-GM1 interactions revealed that the CTB-GM1 binding weakened with increasing GM1 density. Such a result can be explained by the clustering of GM1 on the supported phospholipid membranes, which in turn inhibits the binding of CTB. Chapter IV characterizes the enzymatic activity of phosphatase tethered to SLBs in a microfluidic device. Higher turnover rate and catalytic efficiency were observed at low enzyme surface densities, ascribing to the low steric crowding hindrance and high enzyme fluidity, as well as the resulting improvement of substrate accessibility and affinity of enzyme catalytic sites. Chapter V presents sub-100 nm patterning of supported biomembranes by atomic force microscopy (AFM) based nanoshaving lithography. Stable SLBs formed by this method have a lower size limit of ~ 55 nm in width. This size limit stems from a balance between a favorable bilayer adhesion energy and an unfavorable bilayer edge energy. Finally, chapter VI demonstrates the electrophoretic separation of membrane-associated fluorophores in polymer-cushioned lipid bilayers. This electrophoretic method was applied to the separation of membrane proteins in E. Coli ghost membranes.

Infrared spectroscopy detects molecular vibrations and assesses the properties of molecules and their environment. It is a powerful technique to detect ligand induced changes in biomolecules as it has distinct signals and provides different levels of structural information. An addition of a dialysis accessory to attenuated total reflection infrared spectroscopy makes this technique more universal for ligand binding studies. It facilitates to study ligand binding of substrates, activators, inhibitors and ions to macromolecules as well as effect of pH, ionic strength or denaturants on the structure of macromolecules, which play an important role in drug development. This method was tested with two proteins cyt c and calcium ATPase. We studied phosphoenol pyruvate (PEP) in different ionization states by infrared spectroscopy combined with theoretical analysis. Theoretical calculations helped to assign the bands. The infrared spectrum of labeled PEP and infrared measurement in D2O also helped in band assignment. We used the method dialysis accessory to attenuated total reflection infrared spectroscopy to investigate the binding of PEP and Mg2+ to pyruvate kinase (PK), where conformational changes of PK were revealed upon binding of PEP and Mg2+. Isotopic labeled PEP helped to assign and evaluate the infrared absorption bands. The difference spectrum of bound and free PEP indicates specific interactions between ligand and protein. The quantitative evaluation revealed that the enzyme environment has little influence on the P-O bond strengths, which are weakened by less than 3% upon binding. The carboxylate absorption bands indicate shortening of the C-O bond by as little as 1.3 pm. The binding of PEP to PK in presence of monovalent cations K+ and Na+ showed that the binding interactions are very similar. / doctoral

Hedgehog related genes have been shown to play a major role in development in all deuterostomes. In C.elegans, such genes have been found where the similarity is restricted to the C-terminal domain. This work has focused on the hedgehog related C.elegans proteins called ground (grd), ground-like (grl), and wart (wrt) which appear to form a unique structural family.These proteins are cysteine rich and have conserved cysteine patterns which, together with thethought that they are secreted, are expected to be in disulfide form. Since the extracellular environment is very oxidizing and due to the conserved cysteine pattern, disulfide bonds are thought to play a big part in the folding and stabilization of these proteins. The stability of the protein and the formation of a disulfide bond are related through a thermodynamic cycle, which insures that the stabilization of the protein by the disulfide is reflected by the identical stabilization of the disulfide by the protein. Practically, there are numerous parameters that can be used to try to achieve the correct disulfide bonds and folding, when doing in vitro trials, some of which were used in this project. C.elegans proteins grd-5, grd-13, grl-24, wrt-3 and wrt-5 were studied in this project. All of the proteins were expressed and purified with success, with theexception of grl-24. All constructs formed inclusion bodies. Some refolding attempts were performed on grd-13 and wrt-3. The presence of a disulfide bond in refolded grd-13 was demonstrated using chemical fragmentation. In general, these attempts did not give correctly folded proteins but provide a foundation to continue experiments aimed at producing a native-like protein for structural and functional studies.

Micro/nano-patterning of supported lipid bilayers (SLBs) has shown considerable potential for addressing fundamental biophysical questions about cell membrane behavior and the creation of a new generation of biosensors. Herein are presented several novel lithographic methods for the size-controlled patterning of SLBs from the microscale to the nanoscale. Using these methods, chemically distinct types of phospholipid bilayers and/or Escherichia Coli (E. Coli) membranes can be spatially addressed on a single microchip. These arrays can, in turn, be employed in the studies of multivalent ligand-receptor interactions, enzyme kinetics, SLBs size limitation, and membrane-associated species separation. The investigations performed in the Laboratory for Biological Surface Science include the following projects. Chapters II and III describe the creation of lab-on-a-chip based platforms by patterning SLBs in microfluidic devices, which were employed in high throughput binding assays for multivalent ligand-receptor interactions between cholera toxin B subunits (CTB) and ganglioside GM1. The studies on the effect of ligand density for multivalent CTB-GM1 interactions revealed that the CTB-GM1 binding weakened with increasing GM1 density. Such a result can be explained by the clustering of GM1 on the supported phospholipid membranes, which in turn inhibits the binding of CTB. Chapter IV characterizes the enzymatic activity of phosphatase tethered to SLBs in a microfluidic device. Higher turnover rate and catalytic efficiency were observed at low enzyme surface densities, ascribing to the low steric crowding hindrance and high enzyme fluidity, as well as the resulting improvement of substrate accessibility and affinity of enzyme catalytic sites. Chapter V presents sub-100 nm patterning of supported biomembranes by atomic force microscopy (AFM) based nanoshaving lithography. Stable SLBs formed by this method have a lower size limit of ~ 55 nm in width. This size limit stems from a balance between a favorable bilayer adhesion energy and an unfavorable bilayer edge energy. Finally, chapter VI demonstrates the electrophoretic separation of membrane-associated fluorophores in polymer-cushioned lipid bilayers. This electrophoretic method was applied to the separation of membrane proteins in E. Coli ghost membranes.

Because bimolecular interactions in water are poorly understood, three tactics commonly used to improve binding affinity in ligand design were investigated: (1) increasing apolar surface area, (2) introducing a conformational constraint, and (3) targeting a cation-[pi] interaction. Thermodynamic parameters of binding ligands to the Grb2 SH2 domain were determined by isothermal titration calorimetry (ITC), and structural data was obtained by X-ray crystallography. The apolar surface area of the pTyr+1 residue in Ac-pTyr-Acnc-Asn-NH₂ was varied by incrementally increasing the size of the cyclic Acnc residue from a 3-membered to a 7-membered ring. Increasing apolar surface area resulted in an increase in Ka due to a more favorable [delta]H⁰ that was dominated a less favorable [delta]S⁰. Structural analyses showed that all ligands bound in a similar mode, so differences in binding thermodynamics were attributed to the pTyr+1 residue. The thermodynamics of binding tripeptides wherein pTyr+1 was an n-alkyl group were studied. Ka increased when Ala was mutated to Abu, but additional methylene groups had no effect on Ka due to strong entropy-enthalpy compensation. While [delta]H⁰ was weakly correlated with buried surface area, there was no change in [delta]H⁰ between one methylene and two methylene groups, presumably because an enthalpic penalty is associated with a gauche interaction between C-[beta] and C-[gamma] of the Xaa side chain that was noted in the crystal structure. An olefin was installed in an attempt to alleviate the energetic penalty incurred from the gauche interaction, but the introduction of the constraint resulted in equipotent ligands. A putative cation-[pi] interaction between Arg67 and various aromatic groups was probed by varying the [pi]-donating capability of groups attached to a tripeptide scaffold. Although crystal structures demonstrated that three of the aryl groups were close enough to Arg67 to form a cation-[pi] interaction, only a modest increase in Ka was observed relative to analogues having only an N-acetyl group. Furthermore, a simple cyclohexyl group in place of aryl groups resulted in ligands that were equipotent with indolyl- and phenyl- derived analogues, so any cation-[pi] interaction is not significant. / text

The agroforestry practice of establishing shelterbelts and/or windbreaks composed of tree and shrub species that include buffaloberry (Shepherdia argentea Nutt.), caragana (Caragana arborescens Lam.) and sea buckthorn (Hippophae rhamnoides L.) is widespread within Saskatchewan. Shelterbelts play major roles in reducing wind speed, trapping snow, improving land-use efficiency and increasing economic returns. However, the practice of alley cropping within Saskatchewan is not popular. Also, apart from the protective roles the tree species offer in shelterbelts, some species have atmospheric nitrogen (N2)-fixation capabilities through biological nitrogen fixation (BNF) that are potentially important. The simultaneous integration of trees and crops on the same land management unit may lead to competition between crops and trees for growth resources such as nutrients, soil moisture and incoming radiation, the latter leading to limited access of light for understory crops. Understanding the contributions of the trees in supplying nitrogen (N) through BNF and in modifying microclimatic conditions in the alleyways would generate information needed to know their impacts on yield and nutrition of associated crops. In order to assess the contribution of the tree species in supplying N and minimizing interspecific competition while maximizing the benefits of tree-based intercropping systems, the thesis quantified the BNF capabilities of each species under greenhouse conditions using 15N dilution techniques and assessed how much of the fixed N2 is transferred to associated triticale (Triticale hexaploide Lart.) and oats (Avena sativa L.) under field conditions. Growth and yield of oats was also studied by measuring photosynthetically active radiation (PAR) and soil moisture in a Manitoba maple (Acer negundo var. negundo L.) -oats alley cropping system at Indian Head, SK. The BNF results showed that each of the test species fixes a substantial amount of N and there was a high transfer of N to associated triticale and oats. Results from the interspecific interaction study also showed that soil moisture was the primary factor affecting oats yields followed by light, with the south-lying oat plants affected more than north-lying. It can be concluded that alley cropping systems can be a practical and beneficial agroforestry practice within Saskatchewan. However, the distance between tree rows should be wide enough to permit farm machinery operations.

Precision agriculture (PA) can be defined as a set of technologies that have helped propel agriculture into the computerized information-based world, and is designed to help farmers get greater control over the management of farm operations. Because of its potential to spatially reduce yield variability within the field through variable rate application of nutrients it is thought to be a production risk management instrument. Subsurface drip irrigation (SDI) is another production risk management technology that is generating interest from the farming community as a result of new technological improvements that facilitate equipment maintenance and reduces water consumption.In the first article the production risk management potential of these two technologies was investigated both for each technology and for a combination of the two. Simulated yield data for corn, wheat and soybeans were obtained using EPIC, a crop growth simulation model. Mathematical programming techniques were used in a standard E-V framework to reproduce the production environment of a Kentucky commercial grain farmer in Henderson County. Results show that for risk averse farmers, the lowest yield variability was obtained with the SDI technology. The highest profit level was obtained when the two technologies were combined.Investment in two sets of equipments (PA and SDI) to maximize profitability and reduce risk could however expose many farm operations to financial risk. In the second article, a discrete stochastic sequential programming (DSSP) model was used to analyze the impact of PA and/or SDI equipment investment on the farm's liquidity and debt to asset ratio.In the last article, the cotton sector in Benin, West Africa, was utilized to study the transferability of PA technology to a developing country. Properly introduced, precision agriculture (PA) technology could help farmers increase profitability, improve management practices, and reduce soil depletion. An improved production system could also help farmers better cope with the policy risk related to cotton production. Results from the two models show that PA is less profitable for the risk neutral farmer but more profitable for the risk averse one when compared to conventional production practices. The adoption of the new technology also has very little impact on the choice of crop rotation made by the farmer.

In this research, a recent version of the Boreal Ecosystem Productivity Simulator (BEPS), called BEPS-TerrainLab, was adapted to northern peatlands and evaluated using observations made at the Mer Bleue bog located near Ottawa, Ontario, and the Sandhill fen located near Prince Albert, Saskatchewan. The code was extended and modified with a major focus on the adequate representation of northern peatlands' multi-layer canopy and the associated processes related to energy, water vapour and carbon dioxide fluxes through remotely-sensed leaf area index (LAI) maps. An important prerequisite for the successful mapping of LAI based on remote sensing imagery is the accurate measurement of LAI in the field with a standard technique such as the LAI-2000 plant canopy analyzer. As part of this research, a quick and reliable method to determine shrub LAI with the LAI-2000 instrument was developed. This method was used to collect a large number of LAI data at the Mer Bleue bog for the development of a new remote sensing-based methodology using multiple endmember spectral unmixing that allows for separate tree and shrub LAI mapping in ombrotrophic peatlands. A slight modification of this methodology allows for its application to minerotrophic peatlands and their surrounding landscapes. These LAI maps were used to explicitly represent the tree and shrub layers of the Mer Bleue bog and the tree and shrub/sedge layers of the Sandill fen within BEPS-TerrainLab. The adapted version of BEPS-TerrainLab was used to investigate the in fluence of mescoscale topography (Mer Bleue bog) and macro- and mesoscale topography (Sandhill fen) on wetness, evapotranspiration, and gross primary productivity during the snow-free period of 2004. This research suggests that future peatland ecosystem modelling efforts at regional and continental scales should include a peatland type-specific differentiation of macro- and mesoscale topographic effects on hydrology, to allow for a more realistic simulation of peatlands' soil water balance. This is an important prerequisite for the reduction of currently existing uncertainties in wetlands' contribution to North America's carbon dioxide and methane annual fluxes from an ecosystem modelling perspective.

Made available in DSpace on 2015-07-22T20:50:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-25 / A leptina é uma proteína secretada predominantemente por adipócitos com grande importância na regulação do metabolismo energético em mamíferos. Ela está fortemente envolvida na regulação da obesidade. No hipotálamo, após se ligar, dimerizar e ativar o seu receptor específico, a leptina ativa as quinases JAK que fosforilam fatores de transcrição STAT, que por sua vez induzem um aumento do gasto energético e levam a inibição da fome. A sinalização da leptina é regulada negativamente por uma fosfatase de fosfotirosinas denominada PTP1B. O objetivo deste trabalho é a clonagem, expressão e purificação de leptina humana recombinante em bactéria E. coli. Após indução, a leptina recombinante foi produzida em E.coli BL21(DE3) de forma insolúvel, purificada através de cromatografia em coluna de afinidade de níquel, renovelada através de diálise em série e concentrada a 3 mg/ml. A aplicação desta leptina recombinante em camundongos ob/ob com deficiência de leptina, levou os animais a perderem 8% de seu peso corporal e consumirem 50% menos ração em menos de cinco dias, confirmando a atividade biológica da proteína. Acrescentando o estudo, foi produzida uma mutação dirigida desta leptina recombinante (chamada muteína) com propriedade antagonista. Em paralelo a este trabalho, clonamos, expressamos e purificamos a fosfatase de fosfotirosinas TcPTP1 de Trypanosoma cruzi, visando caracterizar bioquimicamente esta proteína e obter mais informação sobre a função deste homólogo de PTP1B em T.cruzi. A enzima TcPTP1 representa um alvo medicinal interessante por causa de sua possível influencia na regulação das vias de sinalização celular de T.cruzi. Portanto a produção recombinante e caracterização bioquímica realizada da TcPTP1 é a base para elucidar a estrutura e função do domínio catalítico que permitirão o desenho de possíveis inibidores contra esta enzima que aparenta ser vital para a sobrevivência do parasita. / Leptin is predominantly secreted by adipocytes and plays an important role in the regulation of the energetic metabolism of mammals. Because of this, leptin is strongly involved in the regulation of obesity. After ligation, dimerization and activation of its specific receptor in the hypothalamus, leptin activates JAK kinases that phosphorylate STAT transcription factors, which in turn up-regulate energetic expenditure and lead to the inhibition of hunger. Leptin signalling is downregulated by a phosphotyrosine phosphatase named PTP1B. The aim of this work is the cloning, expression and purification of recombinant human leptin in E. coli. After induction, recombinant leptin was produced in E.coli BL21(DE3) in an insoluble form, purified by nickel-afinity chromatography, refolded using serial dialysis and concentrated up to 3mg/ml. Injection of this recombinant protein in ob/ob mice, which are devoid of leptin, leads a reduction of body weight by 8% and a reduction of food intake by 50%, thus confirming the biological activity of the protein. In order to enrich this study, we produced a targeted mutation of this recombinant leptin (called mutein) wich act as an antagonist. In other hand, Additionaly to this work, we also cloned, expressed, purified and biochemically characterized a phosphotyrosine phosphatase from Trypanosoma cruzi termed TcPTP1, which is homologous to mammalian PTP1B. TcPTP1 is an interesting medical target because of its potential influence in the regulation of cell signalling in T.cruzi. The production and biochemical characterization of TcPTP1 is the basis for functional and structural studies that can lead to the design of new inhibitors of this enzyme, which seems to be vital for the parasite. / TEDE / BV UNIFESP: Teses e dissertações