Cis-acting requirements in flanking DNA for the programmed elimination of mse2.9: a common mechanism for deletion of internal eliminated sequences from the developing macronucleus of Tetrahymena thermophila

During macronuclear development in the ciliated protozoan Tetrahymena thermophila, extensive DNA deletions occur, eliminating thousands of internal eliminated sequences (IESs). Using an rDNA-based transformation assay we have analyzed the role during DNA deletion of DNA flanking mse2.9, an IES within the second intron of a gene encoding an as yet incompletely characterized protein. We establish that a cis-acting sequence for mse2.9 deletion acts at a distance to specify deletion boundaries. A complex sequence element necessary for efficient and accurate mse2.9 deletion is located in the region 47–81 bp from the right side of mse2.9. The ability of a variety of IES flanking sequences to rescue a processing deficient mse2.9 construct indicates that some cis-acting signal is shared among different IESs. In addition, the short intronic sequence that flanks mse2.9 is able to direct efficient and accurate processing. Despite no obvious sequence similarity between mse2.9 and other IESs, we suggest that a common mechanism is used to delete different families of IESs in Tetrahymena.

Use of virion DNA as a cloning vector for the construction of mutant and recombinant herpesviruses.

We have developed improved procedures for the isolation of deletion mutant, point mutant, and recombinant herpesvirus saimiri. These procedures take advantage of the absence of NotI and AscI restriction enzyme sites within the viral genome and use reporter genes for the identification of recombinant viruses. Genes for secreted engineered alkaline phosphatase and green fluorescent protein were placed under simian virus 40 early promoter control and flanked by NotI and AscI restriction sites. When permissive cells were cotransfected with herpesvirus saimiri virion DNA and one of the engineered reporter genes cloned within herpesvirus saimiri sequences, recombinant viruses were readily identified and purified on the basis of expression of the reporter gene. Digestion of recombinant virion DNA with NotI or AscI was used to delete the reporter gene from the recombinant herpesvirus saimiri. Replacement of the reporter gene can be achieved by NotI or AscI digestion of virion DNA and ligation with a terminally matched fragment or, alternatively, by homologous recombination in cotransfected cells. Any gene can, in theory, be cloned directly into the virion DNA when flanked by the appropriate NotI or AscI sites. These procedures should be widely applicable in their general form to most or all herpesviruses that replicate permissively in cultured cells.

O português de herança em território fronteiriço: a LH em Olivença como arma para preservação de um grupo minoritário

Esta pesquisa tem como objetivo investigar a língua portuguesa nativa e de herança sobreviventes em território fronteiriço de língua oficial espanhola. Como locus de pesquisa selecionamos Olivença, uma cidade da Espanha em que a língua portuguesa se faz em situações específicas de uso. A relevância desta pesquisa traduz-se no fato de que espaços fronteiriços mantêm sobrepostas em espaços geográficos contíguos algumas realidades que se sobrepõem: a realidade da oficialidade linguística e a realidade do sentimento de pertença sociolinguística. Nesta tese, partimos da identificação, em trabalho de campo, da presença da língua portuguesa no território atualmente espanhol (mas historicamente português) e na constatação de que há uma flutuação de identificação-identidade linguística. No espaço geográfico em que fizemos incursão científica, duas cidades sobrepõem-se historicamente e duas geografias políticas, por outro lado, avizinham-se e roçam-se continuamente. Ao tomar contato com essa cidade, hipotetizamos que o sentimento de pertença linguística estaria presente entre os falantes mais velhos, que manteriam a herança de traços lusitanos em sua comunicação, mesmo ao falar o espanhol. A justificativa é que, logo de chegada, já avistáramos recintos comerciais com nomes portugueses e, contrariamente, não ouvíamos o som lusitano nas ruas. Sabemos que o domínio espanhol numa cidade outrora portuguesa tenderia a apagar vestígios portugueses. No entanto, em grupos íntimos pressupúnhamos o português como língua corrente. Durante o trabalho de campo, identificamos fortes valores culturais sendo empunhados como armas de resistência entre descendentes de portugueses, fazendo correr numa velocidade acentuada a reorganização dos valores lusitanos em redutos da cidade espanhola. Essa força e essa velocidade pareciam ser as molas propulsoras de uma mudança linguística muito sorrateira, que impactava o sentimento de unidade de um segmento social da comunidade sociolinguística. Isso nos inspirou a dar um passo investigativo seguinte em direção aos mais jovens, que tinham o espanhol como língua materna, mas tinham o português como língua de herança. À pergunta central sobre a força do português como língua de herança buscamos respostas por meio de duas outras questões mais indiretas feitas aos sujeitos entrevistados: será que os mais jovens percebiam-se como portugueses? será que os elementos culturais lusitanos presentes nas ruas eram reconhecidos como vinculados à língua de herança? Foi assim que passamos a recolher pistas sobre os traços de resiliência do português como língua incrustada na região espanhola de Olivença.

Fail-Safe Testing of Web Applications

This dissertation introduces an approach to generate tests to test fail-safe behavior for web applications. We apply the approach to a commercial web application. We build models for both behavioral and mitigation requirements. We create mitigation tests from an existing functional black box test suite by determining failure type and points of failure in the test suite and weaving required mitigation based on weaving rules to generate a test suite that tests proper mitigation of failures. A genetic algorithm (GA) is used to determine points of failure and type of failure that needs to be tested. Mitigation test paths are woven into the behavioral test at the point of failure based on failure specific weaving rules. A simulator was developed to evaluate choice of parameters for the genetic algorithm. We showed how to tune the fitness function and performed tuning experiments for GA to determine what values to use for exploration weight and prospecting weight. We found that higher defect densities make prospecting and mining more successful, while lower mitigation defect densities need more exploration. We compare efficiency and effectiveness of the approach. First, the GA approach is compared to random selection. The results show that the GA performance was better than random selection and that the approach was robust when the search space increased. Second, we compare the GA against four coverage criteria. The results of comparison show that test requirements generated by a genetic algorithm (GA) are more efficient than three of the four coverage criteria for large search spaces. They are equally effective. For small search spaces, the genetic algorithm is less effective than three of the four coverage criteria. The fourth coverage criteria is too weak and unable to find all defects in almost all cases. We also present a large case study of a mortgage system at one of our industrial partners and show how we formalize the approach. We evaluate the use of a GA to create test requirements. The evaluation includes choice of initial population, multiplicity of runs and a discussion of the cost of evaluating fitness. Finally, we build a selective regression testing approach based on types of changes (add, delete, or modify) that could occur in the behavioral model, the fault model, the mitigation models, the weaving rules, and the state-event matrix. We provide a systematic method by showing the formalization steps for each type of change to the various models.

Quantum fluctuations stabilize skyrmion textures

We study the quantum spin waves associated to skyrmion textures. We show that the zero-point energy associated to the quantum spin fluctuations of a noncollinear spin texture produce Casimir-like magnetic fields. We study the effect of these Casimir fields on the topologically protected noncollinear spin textures known as skyrmions. In a Heisenberg model with Dzyalonshinkii-Moriya interactions, chosen so the classical ground state displays skyrmion textures, we calculate the spin-wave spectrum, using the Holstein-Primakoff approximation, and the associated zero-point energy, to the lowest order in the spin-wave expansion. Our calculations are done both for the single-skyrmion case, for which we obtain a discrete set of skyrmion bound states, as well as for the skyrmion crystal, for which the resulting spectrum gives the spin-wave bands. In both cases, our calculations show that the Casimir magnetic field contributes up to 10% of the total Zeeman energy necessary to delete the skyrmion texture with an applied field.

What is the Impact of the Underserved Pathway program on Entering an Underserved Family Medicine Residency? A Comparison of Three Approaches for Estimating the Average Treatment Effect

Thesis (Master's)--University of Washington, 2016-06

“Acham que brotamos das fontes dessa cidade?”: uma etnografia sobre o cotidiano de sobrevivência de pessoas em situação de rua em Nata/RN

Through participant observation, held in the administrative region east of Natal / RN, this research aimed to understand about the daily life of men and women living on the streets in the city. How they relate to the space where they live? In this relationship, which uses and survival strategies triggered by this social segment? These were some of the questions that guided this research, in order to highlight the specificities of this way of life and the possible consequences that such a situation could reverberate. In this sense, there was an effort to keep up with people on the streets, their itineraries and seize their narratives. Along the search path - which took place intermittently between the years 2011-2015 - attended spaces of occupation and traffic of this population group in the street, as well as insert me and got involved in events, forums, seminars, meetings and subject of joints on the streets as a political movement (MNPR / RN). They are considered in this study as people on the street to those who occupy the street as their main space of survival and ordering of daily life: in the streets sleep, feed themselves, meet the physiological and hygienic needs, and is where draw sustenance. The street is taken in this research in its broadest sense, including all possible places relatively protected from the cold, rain and exposure to violence, thus includes both open and public spaces: as squares and parks; but also closed and private places: hostels, abandoned warehouses, prisons, etc. It was observed that in none of these spaces guys on the street are established in a fixed manner, in contrast, they experience the roaming, which in part is due to urban systems - which tends to stigmatize them and delete them places - and the very need to survive, because while living in the street differentiated practices are triggered, and these differ from the dominant mode of sedentary life.

“Acham que brotamos das fontes dessa cidade?”: uma etnografia sobre o cotidiano de sobrevivência de pessoas em situação de rua em Nata/RN

Through participant observation, held in the administrative region east of Natal / RN, this research aimed to understand about the daily life of men and women living on the streets in the city. How they relate to the space where they live? In this relationship, which uses and survival strategies triggered by this social segment? These were some of the questions that guided this research, in order to highlight the specificities of this way of life and the possible consequences that such a situation could reverberate. In this sense, there was an effort to keep up with people on the streets, their itineraries and seize their narratives. Along the search path - which took place intermittently between the years 2011-2015 - attended spaces of occupation and traffic of this population group in the street, as well as insert me and got involved in events, forums, seminars, meetings and subject of joints on the streets as a political movement (MNPR / RN). They are considered in this study as people on the street to those who occupy the street as their main space of survival and ordering of daily life: in the streets sleep, feed themselves, meet the physiological and hygienic needs, and is where draw sustenance. The street is taken in this research in its broadest sense, including all possible places relatively protected from the cold, rain and exposure to violence, thus includes both open and public spaces: as squares and parks; but also closed and private places: hostels, abandoned warehouses, prisons, etc. It was observed that in none of these spaces guys on the street are established in a fixed manner, in contrast, they experience the roaming, which in part is due to urban systems - which tends to stigmatize them and delete them places - and the very need to survive, because while living in the street differentiated practices are triggered, and these differ from the dominant mode of sedentary life.

microRNA Regulation of Cellular Immunity

Immunity is broadly defined as a mechanism of protection against non-self entities, a process which must be sufficiently robust to both eliminate the initial foreign body and then be maintained over the life of the host. Life-long immunity is impossible without the development of immunological memory, of which a central component is the cellular immune system, or T cells. Cellular immunity hinges upon a naïve T cell pool of sufficient size and breadth to enable Darwinian selection of clones responsive to foreign antigens during an initial encounter. Further, the generation and maintenance of memory T cells is required for rapid clearance responses against repeated insult, and so this small memory pool must be actively maintained by pro-survival cytokine signals over the life of the host.

T cell development, function, and maintenance are regulated on a number of molecular levels through complex regulatory networks. Recently, small non-coding RNAs, miRNAs, have been observed to have profound impacts on diverse aspects of T cell biology by impeding the translation of RNA transcripts to protein. While many miRNAs have been described that alter T cell development or functional differentiation, little is known regarding the role that miRNAs have in T cell maintenance in the periphery at homeostasis.

In Chapter 3 of this dissertation, tools to study miRNA biology and function were developed. First, to understand the effect that miRNA overexpression had on T cell responses, a novel overexpression system was developed to enhance the processing efficiency and ultimate expression of a given miRNA by placing it within an alternative miRNA backbone. Next, a conditional knockout mouse system was devised to specifically delete miR-191 in a cell population expressing recombinase. This strategy was expanded to permit the selective deletion of single miRNAs from within a cluster to discern the effects of specific miRNAs that were previously inaccessible in isolation. Last, to enable the identification of potentially therapeutically viable miRNA function and/or expression modulators, a high-throughput flow cytometry-based screening system utilizing miRNA activity reporters was tested and validated. Thus, several novel and useful tools were developed to assist in the studies described in Chapter 4 and in future miRNA studies.

In Chapter 4 of this dissertation, the role of miR-191 in T cell biology was evaluated. Using tools developed in Chapter 3, miR-191 was observed to be critical for T cell survival following activation-induced cell death, while proliferation was unaffected by alterations in miR-191 expression. Loss of miR-191 led to significant decreases in the numbers of CD4+ and CD8+ T cells in the periphery lymph nodes, but this loss had no impact on the homeostatic activation of either CD4+ or CD8+ cells. These peripheral changes were not caused by gross defects in thymic development, but rather impaired STAT5 phosphorylation downstream of pro-survival cytokine signals. miR-191 does not specifically inhibit STAT5, but rather directly targets the scaffolding protein, IRS1, which in turn alters cytokine-dependent signaling. The defect in peripheral T cell maintenance was exacerbated by the presence of a Bcl-2YFP transgene, which led to even greater peripheral T cell losses in addition to developmental defects. These studies collectively demonstrate that miR-191 controls peripheral T cell maintenance by modulating homeostatic cytokine signaling through the regulation of IRS1 expression and downstream STAT5 phosphorylation.

The studies described in this dissertation collectively demonstrate that miR-191 has a profound role in the maintenance of T cells at homeostasis in the periphery. Importantly, the manipulation of miR-191 altered immune homeostasis without leading to severe immunodeficiency or autoimmunity. As much data exists on the causative agents disrupting active immune responses and the formation of immunological memory, the basic processes underlying the continued maintenance of a functioning immune system must be fully characterized to facilitate the development of methods for promoting healthy immune function throughout the life of the individual. These findings also have powerful implications for the ability of patients with modest perturbations in T cell homeostasis to effectively fight disease and respond to vaccination and may provide valuable targets for therapeutic intervention.

Conceção de um sistema informático de gestão do pessoal do ISCED – Cabinda

Com um aumento significativo dos dados nos setores empresariais, urge a necessidade de criar formas de facilitar o seu tratamento para um ganho do tempo na execução das atividades, garantindo uma maior eficiência dos sistemas e também proporcionando a maximização do lucro às empresas, quando for esse o caso. O sucesso de uma empresa depende bastante dos seus recursos humanos e estes, constituem o seu “instrumento” mais importante. É (ou seria) das pessoas que a empresa define ações estratégicas, constituindo ganhos de diversas naturezas (de conhecimento, de marca, económicas ou financeiras). O objetivo principal desta dissertação é permitir que a instituição universitária ISCED – Cabinda se adapte ao potencial digital pelo recurso às tecnologias de informação e comunicação, no contexto da sua gestão de Recursos Humanos. Em consequência, a instituição beneficiará com a maior facilidade e rapidez do acesso à informação e a facilidade na atualização dos dados do sistema. Uma vez automatizado, o sistema poderá facilitar o acesso aos dados a fim de responder a uma determinada necessidade que possa existir, independentemente de estar prevista ou não (tornando o sistema mais flexível e adaptável a novas realidades). Com uma Base de dados, far-se-á o cadastro dos dados dos funcionários da instituição fazendo assim evoluir a gestão de Recursos Humanos e proporcionando a busca de informação relativa a um determinado funcionário de uma forma fácil, rápida, precisa, fiável e coerente. Recorrendo ao desenvolvimento de uma aplicação móvel baseada na arquitetura cliente-servidor, as operações de consultas (adicionar, modificar, eliminar), ficam facilitadas e permitem ainda uma maior exploração de dados. A apresentação de uma aplicação móvel para a consulta no âmbito da gestão de Recursos Humanos é complementada com uma agenda de trabalhos a serem desenvolvidos para garantir a proposta de um sistema de gestão de Recursos Humanos para o ISCED.

In silico metabolic modelling of Saccharomyces cerevisiae for the overproduction of succinate using OptKnock and OptGene

Succinate is a naturally occurring metabolite in organism’s cell and is industrially important chemical with various applications in food and pharmaceutical industry. It is also widely used to produce bio-degradable plastics, surfactants, detergents etc. In last decades, emphasis has been given to bio-based chemical production using industrial biotechnology route rather than fossil-based production considering sustainability and environment friendly economy. In this thesis I am presenting a computational model for silico metabolic engineering of Saccharomyces cerevisiae for large scale production of succinate. For metabolic modelling, I have used OptKnock and OptGene optimization algorithms to identify the reactions to delete from the genome-scale metabolic model of S. cerevisiae to overproduce succinate by coupling with organism’s growth. Both OptKnock and OptGene proposed numerous straightforward and non-intuitive deletion strategies when number of constraints including growth constraint to the model were applied. The most interesting strategy identified by both algorithms was deletion combination of pyruvate decarboxylase and Ubiquinol:ferricytochrome c reductase(respiratory enzyme) reactions thereby also suggesting anaerobic fermentation of the organism in glucose medium. Such strategy was never reported earlier for growth-coupled succinate production in S.cerevisiae.

Psychometric properties of the Spanish version of the Coping Strategies Inventory (CSI) in older people

The aim of this study was to evaluate the psychometric properties of the Coping Strategies Inventory (CSI) in a sample of 243 people aged 55 to 99 years old. The first order factorial structure was similar to the structure of the original version of the CSI and to the Spanish adaptation with general population. Furthermore, high levels of internal consistency and of convergent validity were observed in the different primary scales. Nevertheless, the problem avoidance scale did not surpass the validity and reliability tests. Due to it we propose to delete two of the items of this scale. On the other hand, in this study we confirmed the secondary and tertiary structure obtained in the original version of the inventory that was neither confirmed in the Spanish adaptation study nor the other studies on the psychometric properties of the Spanish version of the CSI.

Protecting databases from schema disclosure: a CRUD-based protection model

Database schemas, in many organizations, are considered one of the critical assets to be protected. From database schemas, it is not only possible to infer the information being collected but also the way organizations manage their businesses and/or activities. One of the ways to disclose database schemas is through the Create, Read, Update and Delete (CRUD) expressions. In fact, their use can follow strict security rules or be unregulated by malicious users. In the first case, users are required to master database schemas. This can be critical when applications that access the database directly, which we call database interface applications (DIA), are developed by third party organizations via outsourcing. In the second case, users can disclose partially or totally database schemas following malicious algorithms based on CRUD expressions. To overcome this vulnerability, we propose a new technique where CRUD expressions cannot be directly manipulated by DIAs any more. Whenever a DIA starts-up, the associated database server generates a random codified token for each CRUD expression and sends it to the DIA that the database servers can use to execute the correspondent CRUD expression. In order to validate our proposal, we present a conceptual architectural model and a proof of concept.

Data-stream driven Fuzzy-granular approaches for system maintenance

Intelligent systems are currently inherent to the society, supporting a synergistic human-machine collaboration. Beyond economical and climate factors, energy consumption is strongly affected by the performance of computing systems. The quality of software functioning may invalidate any improvement attempt. In addition, data-driven machine learning algorithms are the basis for human-centered applications, being their interpretability one of the most important features of computational systems. Software maintenance is a critical discipline to support automatic and life-long system operation. As most software registers its inner events by means of logs, log analysis is an approach to keep system operation. Logs are characterized as Big data assembled in large-flow streams, being unstructured, heterogeneous, imprecise, and uncertain. This thesis addresses fuzzy and neuro-granular methods to provide maintenance solutions applied to anomaly detection (AD) and log parsing (LP), dealing with data uncertainty, identifying ideal time periods for detailed software analyses. LP provides deeper semantics interpretation of the anomalous occurrences. The solutions evolve over time and are general-purpose, being highly applicable, scalable, and maintainable. Granular classification models, namely, Fuzzy set-Based evolving Model (FBeM), evolving Granular Neural Network (eGNN), and evolving Gaussian Fuzzy Classifier (eGFC), are compared considering the AD problem. The evolving Log Parsing (eLP) method is proposed to approach the automatic parsing applied to system logs. All the methods perform recursive mechanisms to create, update, merge, and delete information granules according with the data behavior. For the first time in the evolving intelligent systems literature, the proposed method, eLP, is able to process streams of words and sentences. Essentially, regarding to AD accuracy, FBeM achieved (85.64+-3.69)%; eGNN reached (96.17+-0.78)%; eGFC obtained (92.48+-1.21)%; and eLP reached (96.05+-1.04)%. Besides being competitive, eLP particularly generates a log grammar, and presents a higher level of model interpretability.