Monitoring S phase progression globally and locally using BrdU incorporation in TK+ yeast strains

Eukaryotic chromosome replication is initiated from numerous origins and its activation is temporally controlled by cell cycle and checkpoint mechanisms. Yeast has been very useful in defining the genetic elements required for initiation of DNA replication, but simple and precise tools to monitor S phase progression are lacking in this model organism. Here we describe a TK+ yeast strain and conditions that allow incorporation of exogenous BrdU into genomic DNA, along with protocols to detect the sites of DNA synthesis in yeast nuclei or on combed DNA molecules. S phase progression is monitored by quantification of BrdU in total yeast DNA or on individual chromosomes. Using these tools we show that yeast chromosomes replicate synchronously and that DNA synthesis occurs at discrete subnuclear foci. Analysis of BrdU signals along single DNA molecules from hydroxyurea-arrested cells reveals that replication forks stall 8–9 kb from origins that are placed 46 kb apart on average. Quantification of total BrdU incorporation suggests that 190 ‘early’ origins have fired in these cells and that late replicating territories might represent up to 40% of the yeast genome. More generally, the methods outlined here will help understand the kinetics of DNA replication in wild-type yeast and refine the phenotypes of several mutants.

Strategy tools as boundary objects: a strategy-as-practice perspective

This review is structured in three sections and provides a conceptual framework for the empirical analysis of strategy tools as they are used in practice. Examples of strategy tools are SWOT analysis or Porter’s Five Forces, among others. Section one reviews empirical research into the use of strategy tools, classifying them according to variations in their use. Section two explains the concept of boundary objects as the basis for our argument that strategy tools may be understood as boundary objects. Boundary objects are artefacts that are meaningfully and usefully incorporated to enable sharing of information and transfer of knowledge across intra-organizational boundaries, such as laterally across different strategic business units or vertically across hierarchical levels. Section three draws the two bodies of literature together, conceptualizing strategy tools in practice as boundary objects. This review contributes to knowledge on using strategy tools in practice.

A lean kultúra értelmezése és mérése egy egészségügyi szolgáltatónál (The interpretation and measurement of the lean culture – in case of a healthcare service provider)

A lean menedzsment egészségügyi szolgáltatásokra való alkalmazásával elérhető eredmények egyre inkább nyilvánvalóvá válnak. Ennek köszönhetően a szektorban dinamikus növekedés tapasztalható ezen a téren. A kutatások azonban arra hívják fel a figyelmet, hogy a lean menedzsment alkalmazásával elért eredmények csak akkor lesznek fenntarthatóak, ha az eszközök alkalmazását a kultúra átalakulása is követi. A kultúra változásának követéséhez annak folyamatos értékelésére van szükség. A szervezeti kultúra lean-specifikus méréséhez azonban – a szerzők tudomása szerint – még nincs kidolgozott eszköz. Ezért cikkükben a kapcsolódó szakirodalom áttekintése után kidolgoztak egy lean kultúra kérdőívet, majd bemutatják a kérdőív tesztelését és annak eredményeit. Összegzésként elmondható, hogy az itt bemutatott kérdőív az első tesztelés alapján további fejlesztésre szorul. / === / The results that can be obtained by applying lean management in healthcare services become more and more clear. This generates a dynamic increase of lean applications in healthcare. However, researches are warning that the res ults obtained by lean applications can only be sustained, if next to the use of the lean tools cultural change will also take place. In order to track changes in culture its constant evaluation is necessary. According to the authors’ knowledge today does not exist any lean-specific culture evaluation tool. In this paper they elaborate a lean culture questionnaire based on the review of relevant literature. Than they describe its test and the results of the test. The authors conclude that the questionnaire as introduced here needs further improvement.

The role of Lean at the interface with between operations management and applied services within a large aerospace organisation: a boundary spanning perspective

Increased complexity in large design and manufacturing organisations requires improvements at the operations management (OM)–applied service (AS) interface areas to improve project effectiveness. The aim of this paper is explore the role of Lean in improving the longitudinal efficiency of the OM–AS interface within a large aerospace organisation using Lean principles and boundary spanning theory. The methodology was an exploratory longitudinal case approach including exploratory interviews (n = 21), focus groups (n = 2), facilitated action-research workshops (n = 2) and two trials or experiments using longitudinal data involving both OM and AS personnel working at the interface. The findings draw upon Lean principles and boundary spanning theory to guide and interpret the findings. It was found that misinterpretation, and forced implementation, of OM-based Lean terminology and practice in the OM–AS interface space led to delays and misplaced resources. Rather both OM and AS staff were challenged to develop a cross boundary understanding of Lean-based boundary (knowledge) objects in interpreting OM requests. The longitudinal findings from the experiments showed that the development of Lean Performance measurements and lean Value Stream constructs was more successful when these Lean constructs were treated as boundary (knowledge) objects requiring transformation over time to orchestrate improved effectiveness and in leading to consistent terminology and understanding between the OM–AS boundary spanning team.

Criação de estabilidade numa linha de produção de argamassas em pó

Com as constantes mudanças de mercado, as empresas enfrentam cada vez mais obstáculos para se manterem competitivas e inovadoras, para isso procuram formas de sobreviver seguindo filosofias e utilizando ferramentas que permita atingir os seus objetivos. A filosofia Lean é uma filosofia seguida por muitas empresas que pretendam criar valor eliminando desperdícios. O objetivo deste projeto passa pela implementação de ferramentas Lean de modo a criar estabilidade no processo produtivo, eliminando desperdícios e criando valor. Com foco neste objetivo estudou-se as principais fontes de variabilidade no processo produtivo nos 4M’s (Máquina, Método, Mão-de-Obra e Máquina). Após essa análise foram implementadas ações de melhoria como os 5S, criação de sistemas visuais, redução do tempos de setup e tempos de espera, introdução de kanbans e padronização dos métodos de trabalho.Com a implementação destas medidas foi possível tornar o processo produtivo mais estável e flexível, com capacidade para responder às constantes alterações de mercado.

Princípios Lean aplicados à gestão da informação: um caso prático no departamento de custeio da ECCO

O pensamento lean, assim como os conceitos e práticas que lhe estão associados, são, hoje em dia, vistos pelas organizações como meios que promovem a busca pela excelência empresarial. A aplicação dos princípios lean ultrapassou já a barreira do domínio produtivo, conhecendo-se, cada vez mais, outros ramos de utilização, como é o caso do setor dos serviços, que tem demonstrado um potencial enorme no que diz respeito à exploração desta filosofia. No entanto, as experiências de aplicação destes princípios a outras áreas, que não a indústria de transformação, têm ainda poucos resultados concretos espelhados na literatura. Este projeto surge no sentido de contribuir para alcançar processos mais eficientes, aplicando ferramentas e metodologias da filosofia lean a processos de gestão da informação, tendo o caso prático decorrido no recém-criado Departamento de Custeio Global da ECCO. Por se tratar de um novo departamento, ergue-se a necessidade de construir e modificar ferramentas e práticas em toda a estrutura da empresa. Deste modo, o lean será utilizado como um possível caminho para promover a melhoria contínua dos vários processos, tendo em vista a obtenção de uma melhor relação entre os resultados obtidos e os recursos despendidos. Assim, serão apresentadas várias propostas de melhoria para o departamento em causa, no âmbito da gestão da informação e que, partindo de uma análise inicial baseada nos princípios lean, pretendem agregar mais valor aos processos, minimizando os desperdícios que lhes estão associados e, consequentemente, promovendo o seu melhoramento contínuo.

Melhoria de uma linha de montagem pela aplicação da filosofia Lean

Este relatório descreve o desenvolvimento de um projecto de melhoria numa linha de montagem de autocarros, numa das empresas do Grupo Salvador Caetano: a CaetanoBus. Sustentado teoricamente pelos pressupostos da filosofia lean, o projecto visou sobretudo uma organização mais eficiente dos recursos humanos e materiais, de modo a alcançar uma redução do lead time e, por conseguinte, uma diminuição simultânea de desperdícios associados ao processo e do número de colaboradores afectos a determinado modelo de autocarro. A metodologia adoptada envolveu fundamentalmente operações relativas ao estudo de tempos, balanceamento e estabelecimento de standard works. Por outro lado, outros conceitos associados ao lean, como kaizen e yamazum chart, integraram igualmente a linha estruturante de pensamento que orientou o campo das acções. Um plano de melhorias baseado no uso das ferramentas lean sugere ganhos produtivos, muito embora o término da produção do modelo, alvo de análise, tivesse impedido a sua confirmação prática nesta matéria.

Aplicações de Ferramentas Lean em uma Indústria de Transformação de Papel

Esta dissertação foi desenvolvida no âmbito do 2º ano do Mestrado em Engenharia Mecânica – Ramo de Gestão Industrial no Instituto Superior de Engenharia do Porto. Este projeto realizou-se em ambiente industrial, nomeadamente na Tubembal, S.A. uma empresa localizada no concelho da Trofa, distrito do Porto. Esta empresa dedica-se à transformação de papel e comércio de embalagens, produz tubos e cantoneiras de cartão e é atualmente a maior empresa do sector na Península Ibérica. Esta dissertação baseia-se na aplicação de ferramentas Lean, numa perspetiva de melhoria de um ambiente produtivo industrial, melhorando o desempenho dos processos existentes e consequentemente a produtividade da empresa em estudo, com o objetivo de a tornar mais competitiva num ambiente global. A metodologia Lean tem como principal objetivo a eliminação de desperdício em toda a cadeia de valor e neste sentido surge como fundamental numa cultura de melhoria contínua e focalização no cliente, que se pretende instalar nesta empresa. Foi realizada uma análise profunda a toda a cadeia de valor como forma de identificar os maiores desperdícios e posteriormente apresentadas medidas para combater estes mesmos desperdícios, podendo assim reduzir custos. No projeto de melhoria apresentado à organização constam como principais ações, a implementação da metodologia 5S’s como ferramenta essencial para mudança de hábitos dos funcionários e integração e envolvimento de todos num mesmo projeto comum, na busca da melhoria contínua. Procedeu-se ainda à simulação de algumas propostas de reorganização do layout de forma a encontrar aquela que minimizasse os custos com movimentações e garantisse um fluxo controlado e em segurança dos produtos e pessoas dentro da fábrica. As propostas apresentadas mostram que a reorganização do layout da fábrica pode trazer ganhos significativos para a empresa, redução direta no tempo perdido em deslocações e maior disponibilidade dos meios e consequente direta redução dos custos. Todas as propostas apresentadas visam a adaptação da empresa a um modelo mais dinâmico de negócio, capaz de responder rápida e eficazmente aos seus clientes, adaptando-se ao mercado e garantindo a sua sustentabilidade num futuro próximo.

Production of Recombinant Trichoderma Reesei Endoglucanase Protein CEL7B by Using Kluyveromyces Lactis

This research is about producing recombinant Trichoderma reesei endoglucanase Cel7B by using Kluyveromyces lactis, transformed with chromosomally integrated Cel7B cDNA, as a host cell (K. lactis Cel7B). Cel7B is one of the glycoside hydrolyze family of proteins that are produced by T. reesei. Cel7B together with other endoglucanases, exoglucanases, and â-glucosidases hydrolyze cellulose to glucose, which can then be fermented to biofuels or other value-added products. The research objective of this MS project is to examine favorable fermentation conditions for recombinant Cel7B enzyme production and improved activity. Production of enzyme on different types of media was examined, and the activity of the enzyme was measured by using different tools or procedures. The first condition tested for was using different concentrations of galactose as a carbon and energy source; however galactose also acts as a potent promoter of recombinant Cel7B expression in K. lactis Cel7B. The purpose of this method is to determine the relationship between production of enzyme with increasing sugar concentration. The second culture condition test was using different types of media: a complex medium-yeast extract, peptone, galactose (YPGal); a minimal medium-yeast nitrogen base (YNB) with galactose; and a minimal medium with supplement-yeast nitrogen base with casamino acid (YBC), a nitrogen source, with galactose. The third condition was using different types of reactors or fermenters: a small reactor (shake flask) and a larger automated bioreactor (BioFlo 3000 fermenter). The purpose of this method is to determine the quantity of the protein produced by using different environments of production. Different tools to determine the presence and activity of Cel7B enzyme were used. For the presence of enzyme, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used. Secondly, to detect enzyme activity, the carboxymethyl cellulose- 3,5-dinitrosalicylic acid (CMC- DNS) assay was employed. SDS-PAGE showed that the enzyme band was at 67 kDa, which is larger than native Cel7B (52 kDa.), likely due to over glycolylation during post-translational processing in K. lactis. For the different types of media used in our fermentation, recombinant Cel7B was produced from yeast extract peptone galactose (YPGal), and yeast nitrogen base with casamino acid (YBC), but was not produced and no activity was detected from yeast nitrogen base (YNB). This experiment concluded that the Cel7B production requires the amino acid resources as part of fermentation medium. In experiments where recombinant Cel7B net activity was measured at 1% galactose initial concentration in YPGal and YBC media, higher enzyme activity was detected for the complex medium YPGal. Higher activity of recombinant Cel7B was detected for flask culture in 2% galactose compared to 1% galactose for YBC medium. Two bioreactor experiments were conducted under these culture conditions at 30°C, pH 7.0, dissolved oxygen of 50% of saturation, and 250 rpm agitation (variable depending on DO control) K. lactis-Cel7B yeast growth curves were quite reproducible with maximum optical density (O.D) at 600 nm of between 7 and 8 (when factoring dilution of 10:1). Galactose was consumed rapidly during the first 15 hours of bioreactor culture and recombinant Cel7B started to appear in the culture at 10-15 hours and increased thereafter up to a maximum of between 0.9 and 1.6 mg/mL/hr in these experiments. These bioreactor enzyme activity results are much higher than comparable experiments conducted with flask-scale culture (0.5 mg/mL/hr). In order to achieve the highest recombinant Cel7B activity from batch culture of K. lactis-Cel7B, based on this research it is best to use a complex medium, 2% initial galactose concentration, and an automated bioreactor where good control of temperature, pH, and dissolved oxygen can be achieved.

Analysing Road Barrier using GIS Software: The Case Study of Bologna

The direct attention of this thesis is the maintenance of road elements to improve road safety. The goal of the research is to prioritise maintenance for barriers based on factors such as the terrain of the site, deformations, degradation of the components, and adherence to the original installation. Using these factors, a coefficient is calculated to determine the maintenance priority for each barrier. To ease understanding and visualisation, data was uploaded and processed in a GIS environment to generate analysis and maps. This was done using GIS, a free and open-source GIS software. Information about the features of the barriers was collected through both on-site and online examination. During on-site inspections, a database of geotagged photos was created to aid in the survey. GIS capabilities word fully utilised by using geoprocessing tools for more in-depth analysis.

Proposta metodológica utilizando ferramentas de qualidade na avaliação do processo de pulverização

The present work aimed to create a methodology to evaluate the pulverization process with the use of quality tools. It was listed the primary factors, secondary factors, tertiary factors and, with the check list tool support, the list was elaborated. It was evaluated the factors labor, agriculture machine, material and method of 32 pulverization process before pesticide application, in that each factor received a punctuation, having as total sum of 750 points. The medium punctuation to the factors labor, agriculture machine, material and method was 78; 211; 49; 20 and 94 points, respectively. The sum of the factors points for the 32 processes, the minimum value found was 230 and maximum was 620 points. With the proposed methodology, can be identify which common causes of the processes can affect its result.

Planetary transit candidates in Corot-IRa01 field

Context. CoRoT is a pioneering space mission devoted to the analysis of stellar variability and the photometric detection of extrasolar planets. Aims. We present the list of planetary transit candidates detected in the first field observed by CoRoT, IRa01, the initial run toward the Galactic anticenter, which lasted for 60 days. Methods. We analysed 3898 sources in the coloured bands and 5974 in the monochromatic band. Instrumental noise and stellar variability were taken into account using detrending tools before applying various transit search algorithms. Results. Fifty sources were classified as planetary transit candidates and the most reliable 40 detections were declared targets for follow-up ground-based observations. Two of these targets have so far been confirmed as planets, CoRoT-1b and CoRoT-4b, for which a complete characterization and specific studies were performed.

A landscape-scale test of the predictive ability of a spatially explicit model for population viability analysis

1. Although population viability analysis (PVA) is widely employed, forecasts from PVA models are rarely tested. This study in a fragmented forest in southern Australia contrasted field data on patch occupancy and abundance for the arboreal marsupial greater glider Petauroides volans with predictions from a generic spatially explicit PVA model. This work represents one of the first landscape-scale tests of its type. 2. Initially we contrasted field data from a set of eucalypt forest patches totalling 437 ha with a naive null model in which forecasts of patch occupancy were made, assuming no fragmentation effects and based simply on remnant area and measured densities derived from nearby unfragmented forest. The naive null model predicted an average total of approximately 170 greater gliders, considerably greater than the true count (n = 81). 3. Congruence was examined between field data and predictions from PVA under several metapopulation modelling scenarios. The metapopulation models performed better than the naive null model. Logistic regression showed highly significant positive relationships between predicted and actual patch occupancy for the four scenarios (P = 0.001-0.006). When the model-derived probability of patch occupancy was high (0.50-0.75, 0.75-1.00), there was greater congruence between actual patch occupancy and the predicted probability of occupancy. 4. For many patches, probability distribution functions indicated that model predictions for animal abundance in a given patch were not outside those expected by chance. However, for some patches the model either substantially over-predicted or under-predicted actual abundance. Some important processes, such as inter-patch dispersal, that influence the distribution and abundance of the greater glider may not have been adequately modelled. 5. Additional landscape-scale tests of PVA models, on a wider range of species, are required to assess further predictions made using these tools. This will help determine those taxa for which predictions are and are not accurate and give insights for improving models for applied conservation management.

Bioinformatics construction of the human cell surfaceome

Cell surface proteins are excellent targets for diagnostic and therapeutic interventions. By using bioinformatics tools, we generated a catalog of 3,702 transmembrane proteins located at the surface of human cells (human cell surfaceome). We explored the genetic diversity of the human cell surfaceome at different levels, including the distribution of polymorphisms, conservation among eukaryotic species, and patterns of gene expression. By integrating expression information from a variety of sources, we were able to identify surfaceome genes with a restricted expression in normal tissues and/or differential expression in tumors, important characteristics for putative tumor targets. A high-throughput and efficient quantitative real-time PCR approach was used to validate 593 surfaceome genes selected on the basis of their expression pattern in normal and tumor samples. A number of candidates were identified as potential diagnostic and therapeutic targets for colorectal tumors and glioblastoma. Several candidate genes were also identified as coding for cell surface cancer/testis antigens. The human cell surfaceome will serve as a reference for further studies aimed at characterizing tumor targets at the surface of human cells.

The anammox case - A new experimental manifesto for microbiological eco-physiology

The anaerobic ammonium oxidation process is a new process for ammonia removal from wastewater. It is also a new microbial physiology that was previously believed to be impossible. The identification of Candidatus Brocadia anammoxidans and its relatives as the responsible bacteria was only possible with the development of a new experimental approach. That approach is the focus of this paper. The approach is a modernisation of the Winogradsky/Beyerinck strategy of selective enrichment and is based on the introduction of the molecular toolbox and modern bioreactor engineering to microbial ecology. It consists of five steps: (1) postulation of an ecological niche based on thermodynamic considerations and macro-ecological field data; (2) engineering of this niche into a laboratory bioreactor for enrichment culture; (3) black-box physiological characterisation of the enrichment culture as a whole; (4) phylogenetic characterisation of the enriched community using molecular tools; (5) physical separation of the dominant members of the enrichment culture using gradient centrifugation and the identification of the species of interest in accordance with Koch's postulates; (6) verification of the in situ importance of these species in the actual ecosystems. The power of this approach is illustrated with a case study: the identification of the planctomycetes responsible for anaerobic ammonium oxidation. We argue that this was impossible using molecular ecology or conventional 'cultivation based techniques' alone. We suggest that the approach might also be used for the microbiological study of many interesting microbes such as anaerobic methane oxidisers.