Este trabalho teve como objetivo descrever as características sóciodemográficas e de trabalho do profissional da Educação Física da Zona Leste de São Paulo. Como objetivos específicos, identificamos o perfil do profissional e o estilo de vida e saúde, considerando os aspectos de tempo de trabalho, funções exercidas, percepção de satisfação profissional com relação às condições de trabalho, renda e motivação e capacidade para o trabalho do profissional de Educação Física. A metodologia utilizou-se de levantamento bibliográfico, artigos, dissertações e livros, e na utilização de um questionário. Os sujeitos desta pesquisa foram 80 profissionais de Educação Física que atuam nas academias da zona leste de São Paulo. Os itens do questionário foram desmembrados com a finalidade de utilizar os escores pré-definidos de cada instrumento. Foi utilizado o pacote estatístico Statistical Package for the Social Sciences - SPSS 20, estabelecendo uma análise descritiva com cálculos de média, desvio padrão. A maioria dos profissionais era do sexo masculino e tinham média de 33 anos de idade; trabalhavam aproximadamente dois anos na empresa onde foi realizada a pesquisa; dos profissionais, 55 % obtinham de carteira e previdência social. A qualidade de vida e satisfação profissional ficaram na média da classificação boa. Foi identificado uma prevalência baixa de doenças e lesões nestes profissionais. No geral o pentaculo do bem estar teve um resultado positivo para os indicadores de estilo de vida. Podemos afirmar que é uma profissão recente no mercado, e um dos grandes desafios desta área é o processo de envelhecimento e o plano de carreira. Em média esses profissionais tem 2 empregos e dividem a rotina e seu tempo com outras atividades. O gosto pela pratica de atividade física é um fator que colabora para a escolha da profissão em Educação Física. Podemos concluir que os profissionais deste estudo são considerados jovens e fisicamente ativos, além de ter poucos problemas de saúde, uma vez que esses profissionais possuem uma probabilidade maior de cuidar da saúde e ter com o estilo de vida ativo, faz com que os aspectos preventivos sejam mais evidentes / This study aimed to analyze the sociodemographic and work characteristics of the Physical Education professional of the East Zone of São Paulo. As specific objectives, we identify the profile of the professional and aspects of lifestyle and health, considering the aspects of working time, functions exercised, perception of professional satisfaction with regard to working conditions, income and motivation, as well as specify the aptitude for the work of the Physical Education professional. The methodology focused on the bibliographical survey, articles, dissertations and books, and the use of a questionnaire. The subjects of this research were 80 professionals of physical education who work in the academies of the east zone of São Paulo. The items of the questionnaire were dismembered in order to use the pre-defined scores of each instrument. Statistical Package for Social Sciences - SPSS 20 was used, establishing a descriptive analysis with mean, standard deviation calculations. Most of the professionals were male and with an average of 33 years of age; worked for approximately two years in the company where the research was carried out, of the professionals, 55% obtained a portfolio and social security. Quality of life and professional satisfaction were average in the good rating. A low prevalence of diseases and injuries was identified in these professionals. Overall the pentacle of good had a positive outcome for lifestyle indicators. We can state that it is a recent profession in the market, it is one of the great challenges of this area is the aging process and the career plan. On average these professionals have 2 jobs and divide the routine and their time with other activities. The taste for practicing physical activity is a factor that contributes to the choice of the profession in Physical Education. We can conclude that the professionals of this study are considered young and physically active, in addition to having few health problems, since these professionals have a greater probability of taking care of health and having with the active lifestyle, causes that the preventive aspects are more evident

Les machines à commutation de flux cinq-phases présentent une tolérance aux pannes et une robustesse qui les rendent très intéressantes dans un point de vue de la fiabilité, comme montré dans le premier chapitre.Dans ces travaux de thèse nous avons explorés la possibilité de détecter les défauts qui affectent cette machine par la signature des vibrations générées dans la machine.En utilisant les outils physiques et mathématiques présentés dans le deuxième chapitre, nous avons construit deux modèles multiphysiques, un modèle aux les éléments finis développé dans le troisième chapitre et un modèle analytique, appelé aux réseaux de perméances, dans le quatrième chapitre.Le comportement vibratoire de la machine a été étudié à l'aide de ces deux modèles, en régimesain et en défaut afin de connaitre comment ce comportement est influencé par les grandeurs électriques et magnétiques de la machine.Par ailleurs nous avons étudié la possibilité de détecter et discriminer les différents types de défauts.Le modèle analytique se présente comme un bon estimateur du comportement en défaut de la machine, malgré ses écarts avec la simulation.Dans le cinquième chapitre, les deux modèles multiphysiques ont été validés par des essais expérimentaux et nous avons pu expliquer le comportement en défaut d’un point de vue mécanique plutôt que magnétique.Enfin, dans le sixième chapitre, nous avons utilisé les deux modèles pour étudier le comportement en défaut de la machine, à des vitesses au-dessus de la limite expérimentale (3100 tr/min). / Five-phase flux switching machines have a fault tolerance and robustness that makes them very interesting from the point of view of reliability, as shown in chapter one of this work. In our studies we have explored the possibility of detecting faults that affect this type of machine using the signature of stator vibrations.Using the physical and mathematical tools presented in chapter two, we improved two multyphisics models, one based on finite elements method that it's presented in chapter three and the seconde one analitycal model, called permeance networks, in chapter four. The vibratory behavior of the machine was studied using these two models, under healthy and faulty conditions, in order to know how this behavior is influenced by the electrical and magnetic magnitudes of the machine. In addition, we have studied the possibility of detecting and discriminating different types of faults. Analytical model is a good estimator of fault behavior of the machine, despite its differences with the simulation.In chapter five, the two multiphysical models have been validated by experimental tests and we have been able to explain fault behavior by mechanical origin rather than magnetic origin.Finally, in chapter six, we used both models to study the fault behavior of the machine, at speeds above the experimental limit (3100 rpm).

<p>Protein biosynthesis is a core process in all living organisms. Assembly of the protein chain from aminoacids is catalysed by the ribosome, ancient and extremely complex macromolecular machine. Several different classes of accessory molecules are involved in translation, and one set of them, called translational GTPases (trGTPases), was in the focus of this work. </p><p>In this thesis properties of two trGTPases– EF-G and eRF3 - were studied by means of direct biochemical experiments. EF-G is a bacterial trGTPase involved in two steps of translation: translocation and ribosomal recycling. Translocation is a process of the ribosomal movement along the mRNA, and recycling as the step when upon completion of the protein ribosome is released from the mRNA via splitting in two ribosomal subunits. We found that off the ribosome EF-G has similar affinities to GDP and GTP, and thus given the predominance of the latter in the cell, EF-G should be present mostly in the complex with GTP. However, binding to the ribosome increases factors affinity to GTP drastically, ensuring that it is in the GTP-bound state. GDP can not promote neither translocation, not recycling, and GDPNP can not promote recycling. It can, however, promote translocation, but in so doing it results in an intermediate ribosomal state and translocation process can be reversed by addition of GDP, which is not the case for the EF-G•GTP-catalyzed reaction.</p><p>The second trGTPase we investigated is eukaryotic termination factor eRF3. This protein together with another factor, eRF1, is involved translation termination, which is release of the synthesized protein from the ribosome. We demonstrateed, that eRF3 alone has basically no propensity to bind GTP and thus resides in the GDP-bound state. Complex formation between eRF1 and eRF3 promotes GTP binding by the latter, resulting in the formation of the ternary complex eRF1•eRF3•GTP, which in turn is catalyzing the termination event.</p><p>Experimental investigations of trGTPases where rationalized within a generalized thermodynamical framework, accommoding the existent experimental observations, both structural and biochemical. </p>

<p>Higher-order cognitive functions, such as executive function (EF) and intelligence, are crucial to the everyday functioning of human beings. Gaining knowledge about these functions is important for our general understanding of human nature as well as for our ability to help those who may not develop these processes optimally. The present thesis focused particularly on the EF component working memory (WM), described as the ability to maintain informa-tion in consciousness during short time periods with the purpose of using that information in complex cognition. The major aims of the thesis were to increase our understanding of higher-order cognition in children as well as of deficiencies in intelligence found in children with Attention Deficit Hyperactivity Disorder (ADHD). We approached these aims by studying the interrelations among EF-related components in terms of their independent contributions to intellectual functioning. We also studied whether the lower intelligence in children with ADHD was mediated by fundamental EF-related components or whether these deficiencies went beyond the weaknesses in these specific cognitive functions.</p><p>Interpreting the present data, we suggest that intellectual functioning in children is best viewed as representing a system of primarily independent parts that may be accompanied by an overarching common mechanism. The multiple components involve, but are surely not limited to, WM functions, inhibitory functions, sustained attention, and processing speed. One of these functions, WM, was found to be further partitioned into domain-specific executive WM processes and domain-specific short-term storage processes, all of which constitute important aspects of higher-order cognitive functioning. We have further learned that deficits in fluid intelligence in children with ADHD may entail more than weaknesses in specific central cognitive functions. This additional deficit is cautiously interpreted as involving supe-rior executive attention functions setting the stage for the development and integration of the EF system as well as the “intelligence system”.</p>

Systematic calculations were performed to uncover the free energy surfaces for hydrolytic reactions of methyl-diphosphate (in vacuum and implicit solvents) and GTP in EF-Tu active site. Density functional theory and ONIOM extrapolative QM/MM scheme were adopted for the assay. In accordance with experiments, the catalytic effect of the sodium cation was mild. It changes the conformation of GTP attracting its negatively charged oxygen atoms. hydrolýze GTP. The Na+ also equilibrates the charges of all phosphate groups of the GTP mostly by transferring electrons from gamma to beta-phosphate group, which is characteristic for the intermediate states during the hydrolytic reaction.

The Wnt signaling network plays critical roles in development and is implicated in human disease. Wnts comprise a complex signaling network that, upon ligand binding, activates the phosphoprotein Dishevelled (Dvl), leading to distinct outputs including polarized cell movement (known as planar cell polarity, Wnt/PCP) and stabilization of the transcription factor β-catenin (Wnt/β-catenin). The mechanisms that determine a specific output are not completely understood, especially because they share receptors and cellular effectors, such as Naked-cuticle 1 (Nkd), a Dvl-interacting protein. The Nkd protein contains a myristoylation domain and an EF-hand, a putative calcium binding domain. Genetic evidence in Drosophila demonstrates that Nkd acts as a Wnt/β-catenin antagonist, while in contrast, Nkd modulates both branches of Wnt signaling in vertebrates. We hypothesize that the specialized role of Nkd in Drosophila is due to a disrupted EF-hand that cannot not bind calcium. Indeed, this change is unique to Drosophila and is not present in closely related insects all the way up to vertebrates. To test the role of the Nkd EF-hand in Wnt signal integration, we created two different mutations in the zebrafish Nkd: one with a neutralized EF-hand, as well as a Drosophila-like EF-hand, and manipulate Nkd activity in the zebrafish. Using a combination of biochemical and functional assays, we identified a requirement for the Nkd EF-hand in Wnt/PCP but not in Wnt/β-catenin transcriptional outputs. We demonstrate that the Drosophila-like antagonizes Wnt/β-catenin more robustly than zebrafish Nkd. The EF-hand of Nkd is similar to the EF-hand of a known calcium binding protein, Recoverin, a myristoyl-swtich protein that shuttles between the membrane and the cytoplasm depending on its calcium bound state. Consistently, we observe that NkdWT, but not the two mutant forms, shows localization changes in the calcium fluxing cells that also host converging Wnt signals versus calcium quiescent cells. Our functional data suggests that the Nkd EF-hand is important for Wnt signal integration. Interestingly, Nkd only contains one EF-hand, and proteins that bind calcium tend to have multiple. Calcium binding can also be influenced by binding partners. Because of this, we investigate the role of the Nkd binding protein Dvl and their possible calcium affinity. Dvl is a pivotal point in the Wnt signaling network, leading to the output decision of a cell. EF-hand of Nkd binds to the PDZ domain of Dvl. Interestingly, the Dvl PDZ domain contains a region rich in negatively charged amino acids that could aid in binding calcium. In the same manner as Nkd, we generated a Dvl with neutralized putative EF-hand and tested its function and localization relative to wildtype Dvl. This work elucidates the elegant mechanism by which a cell receiving multiple Wnt signals integrates the information into a specific response. The Nkd EF-hand may serve to interpret the physiology of a cell receiving multiple cues and provides mechanistic insight into Wnt signal integration in vivo.

No description available.

The Wnt signaling network has critical roles in development and disease. Simplified, this complex network has two distinct outputs: the Wnt/β-catenin module activates the phosphoprotein Dishevelled (Dvl) and leads to transcriptional activation while the Wnt/Planar Cell Polarity (PCP) module activates Dvl and leads to calcium release and directed cell movement. Wnt/β-catenin and Wnt/PCP share signaling components like Frizzled receptors, Dvl, and Naked (Nkd). It is an open question how converging Wnt signals diverge into separate outcomes. In this thesis, I used molecular techniques, functional studies in the zebrafish, and biochemical approaches to determine the role of Nkd in Wnt signaling. Nkd contains and EF-hand, a putative calcium binding domain, and is known to antagonize Wnt/β-catenin and disrupt Wnt/PCP signaling. We utilized a tissue that requires both Wnt/β-catenin and Wnt/PCP signaling to properly pattern the left/right axes of the embryo; the dorsal forerunner cells (DFCs). The DFCs exhibit aperiodic calcium release as they migrate to form the Kupffer's Vesicle (KV), the organ of asymmetry. Calcium inhibition in the DFCs disrupts their migration, alters KV formation, and disrupts left/right patterning. Nkd is enriched in the DFCs during migration and KV formation and endogenous Nkd knockdown in the DFCs produces the same phenotypes as calcium inhibition, making Nkd a candidate molecule for directing converging Wnt signals to distinct outcomes. To assess the role of the EF-hand in Nkd function, I created point mutations predicted to disrupt EF-hand affinity for calcium. Through functional studies in zebrafish embryos, I determined that Nkd EF-hand is necessary for Nkd function in Wnt/PCP signaling, but dispensable for Wnt/β-catenin signaling. Although Nkd has not been shown to bind calcium, our functional data with the Nkd EF-hand point mutant provides compelling evidence for a role for calcium in Nkd function in directing Wnt signaling output. EF-hand affinity for calcium is influenced by binding partners, and since Nkd binds to Dvl in the Dvl PDZ domain, we screened the domain for a region rich in amino acids that facilitate ion binding. We identified a 12-amino acid sequence in the Dvl PDZ domain with potential to create a negatively charged pocket to help coordinate calcium binding. We expressed the Nkd EF-hand (EFX) Dvl basic domain and PDZ domain (bPDZ). The purified EFX and bPDZ constructs were used to investigate the interaction between Nkd, Dvl, and calcium. I show, by circular dichroism, that the Nkd/Dvl complex undergoes a calcium-induced change in secondary structure. This reveals the mechanism by which Nkd directs Dvl from the default Wnt/β-catenin signaling module to the Wnt/PCP module in response to calcium.

In our efforts to engineer a DNA binding and cleaving protein with greater sequence discrimination, we have designed dimeric proteins derived from engrailed homeodomain and calmodulin. Previous research by our group has shown that a hydrolytically active lanthanide binding site can be incorporated into a DNA binding motif. To understand protein-DNA interaction and improve the sequence selectivity of the chimeric complex, two lanthanide-binding homodimers were designed and expressed. One of the dimers, F2, is coupled together by a flexible polypeptide linker and the other, R7C, is a disulfide cross-linked cysteine mutant at the N-terminus. Studies of fluorescence of tryptophan residues document that the overall affinity for lanthanide and calcium is similar to traditional EF-hand peptides (1-10 μM). Metal titrations monitored by circular dichroism (CD) revealed that the secondary structures of the dimers contained a lower degree of -helicity than the designed monomeric protein due to additional modifications, but because of their flexibility and their two active-site domain, hydrolytic activity was several folds faster than our previously designed proteins and peptides. Unlike earlier reports on our chimeras, F2 also demonstrated the capability to hydrolyze DNA in the presence of some biological relevant metal ions suggesting different cleavage mechanisms were carried out. Extensive DNA sequencing studies on cleavage patterns with oligonucleotide duplexes confirmed the unique sequence selectivity and kinetic properties of F2. Two engrailed homeodomain target sites, TAATTA, were favored for hydrolytic activity corresponding to one domain acting as a DNA anchor on the first target site while the other was an "opportunist" at recognizing the second site. Nonetheless, the hydrolytic behavior at the phosphodiester bond on a specific dsDNA sequence is in good agreement with the behavior of restriction endonucleases. Unlike restriction enzymes, metallated F2 has not only demonstrated the ability to cleave DNA plasmid, but it also excises the entire nucleotide on a selected sequence. This homodimer is the first example of an active and selective hydrolytic artificial nuclease based on the modular turn substitution design approach that can be a potential template for genomic modification.

Protein biosynthesis is a core process in all living organisms. Assembly of the protein chain from aminoacids is catalysed by the ribosome, ancient and extremely complex macromolecular machine. Several different classes of accessory molecules are involved in translation, and one set of them, called translational GTPases (trGTPases), was in the focus of this work. In this thesis properties of two trGTPases– EF-G and eRF3 - were studied by means of direct biochemical experiments. EF-G is a bacterial trGTPase involved in two steps of translation: translocation and ribosomal recycling. Translocation is a process of the ribosomal movement along the mRNA, and recycling as the step when upon completion of the protein ribosome is released from the mRNA via splitting in two ribosomal subunits. We found that off the ribosome EF-G has similar affinities to GDP and GTP, and thus given the predominance of the latter in the cell, EF-G should be present mostly in the complex with GTP. However, binding to the ribosome increases factors affinity to GTP drastically, ensuring that it is in the GTP-bound state. GDP can not promote neither translocation, not recycling, and GDPNP can not promote recycling. It can, however, promote translocation, but in so doing it results in an intermediate ribosomal state and translocation process can be reversed by addition of GDP, which is not the case for the EF-G•GTP-catalyzed reaction. The second trGTPase we investigated is eukaryotic termination factor eRF3. This protein together with another factor, eRF1, is involved translation termination, which is release of the synthesized protein from the ribosome. We demonstrateed, that eRF3 alone has basically no propensity to bind GTP and thus resides in the GDP-bound state. Complex formation between eRF1 and eRF3 promotes GTP binding by the latter, resulting in the formation of the ternary complex eRF1•eRF3•GTP, which in turn is catalyzing the termination event. Experimental investigations of trGTPases where rationalized within a generalized thermodynamical framework, accommoding the existent experimental observations, both structural and biochemical.