Mechanical behavior in living cells consistent with the tensegrity model

Alternative models of cell mechanics depict the living cell as a simple mechanical continuum, porous filament gel, tensed cortical membrane, or tensegrity network that maintains a stabilizing prestress through incorporation of discrete structural elements that bear compression. Real-time microscopic analysis of cells containing GFP-labeled microtubules and associated mitochondria revealed that living cells behave like discrete structures composed of an interconnected network of actin microfilaments and microtubules when mechanical stresses are applied to cell surface integrin receptors. Quantitation of cell tractional forces and cellular prestress by using traction force microscopy confirmed that microtubules bear compression and are responsible for a significant portion of the cytoskeletal prestress that determines cell shape stability under conditions in which myosin light chain phosphorylation and intracellular calcium remained unchanged. Quantitative measurements of both static and dynamic mechanical behaviors in cells also were consistent with specific a priori predictions of the tensegrity model. These findings suggest that tensegrity represents a unified model of cell mechanics that may help to explain how mechanical behaviors emerge through collective interactions among different cytoskeletal filaments and extracellular adhesions in living cells.

Real-time detection of the surface delivery of newly synthesized membrane proteins.

Newly synthesized membrane proteins travel from the Golgi complex to the cell surface in transport vesicles. We have exploited the ion channel properties of the nicotinic acetylcholine receptor (AChR) to observe in real time the constitutive delivery of newly synthesized AChR proteins to the plasma membrane in cultured muscle cells. Whole-cell voltage clamp was employed to monitor the current fluctuations induced by carbamylcholine upon the insertion into the plasma membrane of newly synthesized AChRs, following release from a 20 degrees C temperature block. We find that the transit of vesicles to the cell surface occurs within a few minutes after release of the block. The time course of electrical signals is consistent with many of the fusion events being instantaneous, although some appear to reveal the flickering of a fusion pore. AChR-containing vesicles can fuse individually or as conglomerates. Intracellular application of guanosine 5'-[gamma-thio]triphosphate inhibits the constitutive traffic of AChRs in most cells. Individual exocytotic vesicles carry between 10 and 300 AChR molecules, suggesting that AChRs may be packed extremely densely.

Probing the oligomeric structure of an enzyme by electrospray ionization time-of-flight mass spectrometry.

Electrospray ionization time-of-flight (ESI-TOF) mass spectrometry was used to study the quaternary structure of 4-oxalocrotonate tautomerase (EC 5.3.2; 4OT), and four analogues prepared by total chemical synthesis. Wild-type 4OT is a hexamer of 62 amino acid subunits and contains no cysteine residues. The analogues were: (desPro1)4OT, a truncated construct in which Pro1 was deleted; (Cpc1)4OT in which Pro1 was replaced with cyclopentane carboxylate; a derivative [Met(O)45]4OT in which Met45 was oxidized to the sulfoxide; and an analogue (Nle45)4OT in which Met45 was replaced with norleucine. ESI of (Nle45)4OT, (Cpc1)4OT, and 4OT from solution conditions under which the native enzyme was fully active (5 mM ammonium bicarbonate buffer, pH 7.5) gave the intact hexamer as the major species detected by TOF mass spectrometry. In contrast, analysis of [Met(O)45]4OT and (desPro1)4OT under similar conditions yielded predominantly monomer ions. The ESI-TOF measurements were consistent with structural data obtained from circular dichroism spectroscopy. In the context of kinetic data collected for 4OT and these analogues, ESI-TOF mass spectrometry also provided important evidence for the structural and mechanistic significance of the catalytically important Pro1 residue in 4OT.

Stopped-flow NMR spectroscopy: real-time unfolding studies of 6-19F-tryptophan-labeled Escherichia coli dihydrofolate reductase.

Escherichia coli dihydrofolate reductase (DHFR; EC 1.5.1.3) contains five tryptophan residues that have been replaced with 6-19F-tryptophan. The 19F NMR assignments are known in the native, unliganded form and the unfolded form. We have used these assignments with stopped-flow 19F NMR spectroscopy to investigate the behavior of specific regions of the protein in real time during urea-induced unfolding. The NMR data show that within 1.5 sec most of the intensities of the native 19F resonances of the protein are lost but only a fraction (approximately 20%) of the intensities of the unfolded resonances appears. We postulate that the early disappearance of the native resonances indicates that most of the protein rapidly forms an intermediate in which the side chains have considerable mobility. Stopped-flow far-UV circular dichroism measurements indicate that this intermediate retains native-like secondary structure. Eighty percent of the intensities of the NMR resonances assigned to the individual tryptophans in the unfolded state appear with similar rate constants (k approximately 0.14 sec-1), consistent with the major phase of unfolding observed by stopped-flow circular dichroism (representing 80% of total amplitude). These data imply that after formation of the intermediate, which appears to represent an expanded structural form, all regions of the protein unfold at the same rate. Stopped-flow measurements of the fluorescence and circular dichroism changes associated with the urea-induced unfolding show a fast phase (half-time of about 1 sec) representing 20% of the total amplitude in addition to the slow phase mentioned above. The NMR data show that approximately 20% of the total intensity for each of the unfolded tryptophan resonances is present at 1.5 sec, indicating that these two phases may represent the complete unfolding of the two different populations of the native protein.

Real-time detection of DNA hybridization and melting on oligonucleotide arrays by using optical wave guides.

The challenge of the Human Genome Project is to increase the rate of DNA sequence acquisition by two orders of magnitude to complete sequencing of the human genome by the year 2000. The present work describes a rapid detection method using a two-dimensional optical wave guide that allows measurement of real-time binding or melting of a light-scattering label on a DNA array. A particulate label on the target DNA acts as a light-scattering source when illuminated by the evanescent wave of the wave guide and only the label bound to the surface generates a signal. Imaging/visual examination of the scattered light permits interrogation of the entire array simultaneously. Hybridization specificity is equivalent to that obtained with a conventional system using autoradiography. Wave guide melting curves are consistent with those obtained in the liquid phase and single-base discrimination is facile. Dilution experiments showed an apparent lower limit of detection at 0.4 nM oligonucleotide. This performance is comparable to the best currently known fluorescence-based systems. In addition, wave guide detection allows manipulation of hybridization stringency during detection and thereby reduces DNA chip complexity. It is anticipated that this methodology will provide a powerful tool for diagnostic applications that require rapid cost-effective detection of variations from known sequences.

Relações entre família, políticas de assistência social e vulnerabilidade social: um estudo a partir de entrevistas com mulheres usuárias do SUAS

A centralidade na família é uma das principais diretrizes adotadas pela Política Nacional de Assistência Social (PNAS) implementada por meio do Sistema Único de Assistência Social (SUAS). A noção de matricialidade sociofamiliar configura-se como base para a formulação de serviços específicos de abordagem familiar nos níveis de proteção social básica e especial, que visam o acompanhamento longitudinal de famílias em situação de vulnerabilidade social, definida neste contexto pela condição de desvantagem em decorrência da pobreza, da privação de renda e de acesso aos serviços e bens públicos, da fragilização de vínculos afetivos, relacionais e de pertencimento. Objetivou-se com esse estudo identificar como as mulheres usuárias do SUAS percebiam e definiam as vulnerabilidades do próprio núcleo familiar, assim como verificar se a concepção de vulnerabilidade social proposta pela PNAS é condizente com as problemáticas apresentadas pelas famílias usuárias do SUAS. A pesquisa qualitativa foi executada por meio de entrevistas de longa duração com temas advindos da observação participante realizada ao longo de três anos de acompanhamento de mulheres usuárias do SUAS por meio de abordagem grupal em serviço de Assistência Social do bairro do Butantã na cidade de São Paulo. Participaram do estudo oito mulheres, acima de 18 anos, usuárias do SUAS e residentes da zona oeste de São Paulo. As depoentes têm entre 35 e 51 anos de idade, estudaram entre a 5ª e a 8ª série do Ensino Fundamental II, cinco delas são migrantes, quatro vivem com os companheiros e filhos e quatro residem com os filhos em uma organização familiar monoparental, três são beneficiárias da Previdência Social, duas estão inseridas em trabalhos formais, duas em trabalhos informais e uma não trabalha. Todas relataram ter sofrido episódios de violência e violação de direitos, principalmente observados na dificuldade de acesso a serviços sociais básicos. Os depoimentos marcaram dificuldades experimentadas ao longo da vida das mulheres e a tônica dos relatos destacou as preocupações atreladas ao papel de mãe e à luta cotidiana que enfrentam para educar, orientar, sustentar e estar junto aos filhos, sobretudo daqueles que se encontram na fase da infância e da adolescência. À luz da noção de enraizamento, proposta por Simone Weil, foi possível identificar que as depoentes apresentam e enfrentam cotidianamente dificuldades ligadas ao desenraizamento urbano, elucidado especialmente na carência de participação comunitária e política. Nesse cenário, a condição de vulnerabilidade pela qual as famílias são definidas no âmbito da política de assistência social, revela certa ambiguidade, pois ao mesmo tempo em que permite oferecer a essas famílias alguma modalidade de apoio também as coloca numa posição estigmatizada de beneficiárias das políticas públicas. Com isso, considerou-se que apesar da concepção de vulnerabilidade proposta pela PNAS ser condizente com as problemáticas apresentadas pelas usuárias, verifica-se uma lacuna em relação à dimensão subjetiva da formação de cada grupo familiar e suas necessidades específicas a fim de apoiá-los para a promoção de uma mudança efetiva

Chinese Intellectuals and China`s Policy toward Japan

This dissertation aims at integrating two scholarships: state-society relation studies and Chinese foreign policy analysis. I created Two-level Perception Gap Model to analyze different intellectual groups' relations with party-state by confirming Chinese intellectuals play a role in CFP making in general, China's Japan policy in particular. This model is an alternative approach, instead of conventional wisdom patron-client approach, to explain and analyze the pluralized intellectual-state relations in China. This model first analyzed the role of two intellectual groups, namely think tank scholars and popular nationalist, in China's Japan policy making, and then based on these analyses it explains the interactional patterns between these two intellectual groups and party-state. I used three case studies, which represented different types of issue, Chinese attitude toward the U.S.-Japan alliance and the Japanese defense policy, the controversy over the Yasukuni Shrine Visit, and the territorial dispute over the Diaoyu/Senkaku Islands, to examine this model. First, I examined think tank scholar groups and the extent they influenced "core interest issue and sensitive issue (Issue 1)," Chinese attitude toward the U.S.-Japan alliance and the Japanese defense policy, and their international patterns with party-state. Chapter 3 compares the responses of Chinese officials to the changes in the defense policy of Japan to the analyses from the think tank scholars. As the model assumes, results show that think tank scholars' analyses are consistent with China's policy position; nevertheless, it is difficult to confirm their analyses have influence on Chinese attitude toward the U.S.-Japan alliance and the Japanese defense policy. Based on the analysis of journal articles, most articles do not provide policy suggestions or simply provide suggestions that do not deviate from the policy. As Gu's theory of pluralist institutionalism and my hypothesis points out, most think tank scholars are establishment intellectuals so they tend to be self-disciplined. Second, this model provide a new concept "patriotic dilemma" for analyzing the challenge and constraints brought by popular nationalist discourses and public mobilization to Chinese foreign policy decision makers. Chapter 4 investigated the cases study of the controversy over the Yasukuni Shrine Visit, defined as "major/minor interest issue/ sensitive issue (Issue 3)," and the discourses from the popular nationalist, mainly focusing on anti-Japanese activists. The chapter also observes their influence on nationalist public opinions and analyzes how the nationalist public opinions constrain the policy choices among decision makers. Results strongly supported the hypothesis of patriotic dilemma that, although the popular nationalist group and public opinions constrained the policy choices of Chinese decision makers in the short term, they were unable to change the fundamental policy direction. Third, chapter 5 also focuses on anti-Japanese activists and examines the model with the case of the territorial dispute over the Diaoyu/Senkaku Islands. The result supported that hypothesis that China's policy change was not because of the influence from popular nationalist's discourses or public opinions but because of the change of priority of this issue, from major/minor interest issue to core interest issue. These two chapters also indicate that the patron-client model is unable to describe the popular nationalist. An alternative approach, such as the concept "patriotic dilemma" is needed to describe the relations between the popular nationalist and the government.

Extended theory about the allocation of time: Description and application to the increase in the retirement age policies

This paper revises mainstream economic models which include time use in an explicit and endogenous manner, suggesting a extended theory which escape from the main problem existing in the literature. In order to do it, we start by presenting in section 2 the mainstream time use models in economics, showing their main features. Once this is done, we introduce the reader in the main problems this kind of well established models imply, within section 3, being the most highlighted the problem of joint production. Subsequently, we propose an extended theory which solves the problem of joint production; this is extensively described in section 4. Last, but not least, we apply this model to offer a time use analysis of the effect of a policy which increases the retirement age in a life-cycle perspective for a representative individual.

Real time optimization in chemical process: evaluation of strategies, improvements and industrial application.

The increasing economic competition drives the industry to implement tools that improve their processes efficiencies. The process automation is one of these tools, and the Real Time Optimization (RTO) is an automation methodology that considers economic aspects to update the process control in accordance with market prices and disturbances. Basically, RTO uses a steady-state phenomenological model to predict the process behavior, and then, optimizes an economic objective function subject to this model. Although largely implemented in industry, there is not a general agreement about the benefits of implementing RTO due to some limitations discussed in the present work: structural plant/model mismatch, identifiability issues and low frequency of set points update. Some alternative RTO approaches have been proposed in literature to handle the problem of structural plant/model mismatch. However, there is not a sensible comparison evaluating the scope and limitations of these RTO approaches under different aspects. For this reason, the classical two-step method is compared to more recently derivative-based methods (Modifier Adaptation, Integrated System Optimization and Parameter estimation, and Sufficient Conditions of Feasibility and Optimality) using a Monte Carlo methodology. The results of this comparison show that the classical RTO method is consistent, providing a model flexible enough to represent the process topology, a parameter estimation method appropriate to handle measurement noise characteristics and a method to improve the sample information quality. At each iteration, the RTO methodology updates some key parameter of the model, where it is possible to observe identifiability issues caused by lack of measurements and measurement noise, resulting in bad prediction ability. Therefore, four different parameter estimation approaches (Rotational Discrimination, Automatic Selection and Parameter estimation, Reparametrization via Differential Geometry and classical nonlinear Least Square) are evaluated with respect to their prediction accuracy, robustness and speed. The results show that the Rotational Discrimination method is the most suitable to be implemented in a RTO framework, since it requires less a priori information, it is simple to be implemented and avoid the overfitting caused by the Least Square method. The third RTO drawback discussed in the present thesis is the low frequency of set points update, this problem increases the period in which the process operates at suboptimum conditions. An alternative to handle this problem is proposed in this thesis, by integrating the classic RTO and Self-Optimizing control (SOC) using a new Model Predictive Control strategy. The new approach demonstrates that it is possible to reduce the problem of low frequency of set points updates, improving the economic performance. Finally, the practical aspects of the RTO implementation are carried out in an industrial case study, a Vapor Recompression Distillation (VRD) process located in Paulínea refinery from Petrobras. The conclusions of this study suggest that the model parameters are successfully estimated by the Rotational Discrimination method; the RTO is able to improve the process profit in about 3%, equivalent to 2 million dollars per year; and the integration of SOC and RTO may be an interesting control alternative for the VRD process.

Paris, France, ca. 4th Century (Time Series Map 1 of 8) (Raster Image)

This layer is a georeferenced raster image from the historic paper map series entitled: Lutece ... plan de la ville de Paris ..., par M.L.C.D.L.M. ; A. Coquart, delineavit et sculp. It was published by Jean & Pierre Cot in 1705. Scale [ca. 1:10,000]. This image is of map 1 entitled: Lutece, ou, premier plan de la ville de Paris: tiré de Cesar, de Strabon, de l'empereur Iulien, et d'Ammian Marcellin. The map represents Paris ca. 4th Century. Map in French.The image inside the map neatline is georeferenced to the surface of the earth and fit to the European Datum 1950, Universal Transverse Mercator (UTM) Zone 31N projected coordinate system. All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, index maps, legends, or other information associated with the principal map. This map shows features such as drainage, towns and villages, roads, selected buildings, ground cover, and more. Relief shown by hachures. This layer is part of a selection of digitally scanned and georeferenced historic maps from the Harvard Map Collection. These maps typically portray both natural and manmade features. The selection represents a range of originators, ground condition dates, scales, and map purposes.

Paris, France, 5th-10th Century (Time Series Map 2 of 8) (Raster Image)

This layer is a georeferenced raster image from the historic paper map series entitled: Lutece ... plan de la ville de Paris ..., par M.L.C.D.L.M. ; A. Coquart, delineavit et sculp. It was published by Jean & Pierre Cot in 1705. Scale [ca. 1:10,000]. This image is of map 2 entitled: Lutece conquise par les François sur les Romains, ou, second plan de la ville de Paris: tiré du misopogone de l'empereur Iulien, d'Amian Marcellin, de Gregoire de Tours, de Boece, de l'abbé Suger, des ancients titres, du tresor des chartres, des archives de St. Germain, de St. Magloire, de St. Eloy, du temple et des vestiges de cette ancienne enceinte qui subsiste encore au jourd'huy ou l'on a vû de nos jours. The map represents Paris, 5th to 10th Centuries. Map in French.The image inside the map neatline is georeferenced to the surface of the earth and fit to the European Datum 1950, Universal Transverse Mercator (UTM) Zone 31N projected coordinate system. All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, index maps, legends, or other information associated with the principal map. This map shows features such as drainage, towns and villages, roads, built-up areas and selected buildings, fortification, ground cover, and more. Relief shown by hachures.This layer is part of a selection of digitally scanned and georeferenced historic maps from the Harvard Map Collection. These maps typically portray both natural and manmade features. The selection represents a range of originators, ground condition dates, scales, and map purposes.

Paris, France, 1137-1180 (Time Series Map 3 of 8) (Raster Image)

This layer is a georeferenced raster image from the historic paper map series entitled: Lutece ... plan de la ville de Paris ..., par M.L.C.D.L.M. ; A. Coquart, delineavit et sculp. It was published by Jean & Pierre Cot in 1705. Scale [ca. 1:10,000]. This image is of map 3 entitled: Troisieme plan de la ville de Paris son êtendiie et les bourgs dont elle êtoit environnée sous le regne de Louis le Jeune VIIe du nom: tiré des descriptions de Fortunat, de Gregoire de Tours, Dabbon, et D'Aimoin, et des anciens titres qui sont dans les Tresor des chartres de France, les registres de la Chambre, des Comptes, les Banieres du Châtelet de Paris, les Archives de l'Archevêché celles de Saint Germain, de Ste. Genevieve, de St. Martin, de S. Eloy, et du Temple. The map represents Paris, 1137 to 1180. Map in French.The image inside the map neatline is georeferenced to the surface of the earth and fit to the European Datum 1950, Universal Transverse Mercator (UTM) Zone 31N projected coordinate system. All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, index maps, legends, or other information associated with the principal map. This map shows features such as drainage, towns and villages, roads, built-up areas and selected buildings, fortification, ground cover, and more. Relief shown by hachures.This layer is part of a selection of digitally scanned and georeferenced historic maps from the Harvard Map Collection. These maps typically portray both natural and manmade features. The selection represents a range of originators, ground condition dates, scales, and map purposes.

Paris, France, 1180-1223 (Time Series Map 4 of 8) (Raster Image)

This layer is a georeferenced raster image from the historic paper map series entitled: Lutece ... plan de la ville de Paris ..., par M.L.C.D.L.M. ; A. Coquart, delineavit et sculp. It was published by Jean & Pierre Cot in 1705. Scale [ca. 1:10,000]. This image is of map 4 entitled: Quatriême plan de la ville de Paris son accroissement, et l'etat ou elle êtoit sous le regne de Philippe Auguste qui mourut l'an 1223 apres avoir regné 43 ans: tiré de Rigord, de Knobelsderf, de Rodolphe Boterée, de Raoul, de Praesles, de Paul Merula, de Guaquin, de Pithou, de Papire Masson, de Corrozet, de Dubreüil, de Duchesne, des memoriaux, et autres anciens registres de la Chambre, des Comptes, et des archives de l'Archevêché du Chaptire de N. Dame, et des anciennes abbayes. The map represents Paris, 1180 to 1223. Map in French.The image inside the map neatline is georeferenced to the surface of the earth and fit to the European Datum 1950, Universal Transverse Mercator (UTM) Zone 31N projected coordinate system. All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, index maps, legends, or other information associated with the principal map. This map shows features such as drainage, towns and villages, roads, built-up areas and selected buildings, fortification, ground cover, and more. Relief shown by hachures. Includes index.This layer is part of a selection of digitally scanned and georeferenced historic maps from the Harvard Map Collection. These maps typically portray both natural and manmade features. The selection represents a range of originators, ground condition dates, scales, and map purposes.

Paris, France, 1367-1383 (Time Series Map 5 of 8) (Raster Image)

This layer is a georeferenced raster image from the historic paper map series entitled: Lutece ... plan de la ville de Paris ..., par M.L.C.D.L.M. ; A. Coquart, delineavit et sculp. It was published by Jean & Pierre Cot in 1705. Scale [ca. 1:10,000]. This image is of map 5 entitled: Cinquiême plan de la ville de Paris son accroissement et sa quatriême clôture commancée sous Charles V. l'an 1367 et finie sous Charles VI. l'an 1383: tiré des devis et marchez faits avec le ouvriers, des procez verbaux de toisez et receptions des ouvrages des comptes rendus par ceux qui en eurent la conduite, de la chronique m.s. de St. Denis et d'autres titres et manuscrits qui sont conservez en la Chambre des Comptes et dans Biblioteques. The map represents Paris, 1367 to 1383. Map in French.The image inside the map neatline is georeferenced to the surface of the earth and fit to the European Datum 1950, Universal Transverse Mercator (UTM) Zone 31N projected coordinate system. All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, index maps, legends, or other information associated with the principal map. This map shows features such as drainage, towns and villages, roads, built-up areas and selected buildings, fortification, ground cover, and more. Relief shown by hachures. Includes index, text, and notes.This layer is part of a selection of digitally scanned and georeferenced historic maps from the Harvard Map Collection. These maps typically portray both natural and manmade features. The selection represents a range of originators, ground condition dates, scales, and map purposes.

Paris, France, 1589-1643 (Time Series Map 7 of 8) (Raster Image)

This layer is a georeferenced raster image from the historic paper map series entitled: Lutece ... plan de la ville de Paris ..., par M.L.C.D.L.M. ; A. Coquart, delineavit et sculp. It was published by Jean & Pierre Cot in 1705. Scale [ca. 1:10,000]. This image is of map 7 entitled: Septiême plan de la ville de Paris son acroissement [sic] et ses embelissemens sous Henry III. et Louis XIII. depuis 1589 jusqu'en 1643: tiré des lettres patentes ou arrest du conseil qui ont ordonné les ouvrages des devis et marchez faits avec les entrepreneurs et levé sur les lieux ou ils ont été construits, et ou la plus grand partie subsistent encore. The map represents Paris, 1589 to 1643. Map in French.The image inside the map neatline is georeferenced to the surface of the earth and fit to the European Datum 1950, Universal Transverse Mercator (UTM) Zone 31N projected coordinate system. All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, index maps, legends, or other information associated with the principal map. This map shows features such as drainage, towns and villages, roads, built-up areas and selected buildings, fortification, ground cover, and more. Relief shown by hachures. Includes index, text, and notes.This layer is part of a selection of digitally scanned and georeferenced historic maps from the Harvard Map Collection. These maps typically portray both natural and manmade features. The selection represents a range of originators, ground condition dates, scales, and map purposes.