Tuotannonsuunnittelun analysointi ja kehittäminen eräällä sahalaitoksella

Diplomityön tavoitteena oli analysoida ja kehittää työn toimeksiantajan UPM-Kymmene Wood Oy Korkeakosken sahan tuotannonsuunnittelua. Työn tärkein tavoite on saada selkeä toimintamalli tuotannonsuunnitteluun, jonka avulla pystytään läpäisemään 300 000 m3:n tai jopa tulevaisuudessa 320 000 m3:n vuosituotanto. Tuotannonsuunnittelulle asetettavat perusvaatimukset ovat raaka-aineen, tuotannon ja myynnin yhteensovittaminen, tilausten oikea-aikainen valmistus ja toimitus, asiakastyytyväisyys, laadun tasaisuus, reklamaatioiden minimointi ja ajosarjojen pituuden maksimointi. Tuotannon suunnittelussa on tärkeää tuntea raaka-aine- ja tuotejakaumat, varastot sekä materiaalivirrat. Tärkeitä osa-alueita tässä tutkimuksessaovat raaka-aine- sekä väli- ja valmisvarastojen seuranta, kierto sekä tuotannonohjaus. Tutkimuksessa perehdytään tuotantoon ja tuotannonsuunnittelun nykyiseen toimintaan ja apuvälineisiin. Tutkimuksen tuloksena kehitettiin tuotannonsuunnitteluun toimintamalli. Se vaatii tuotannonsuunnittelijalta tarkkaa analysointia ja tuotteiden läpimenoaikojen tuntemusta sekä kuivauksen maksimikapasiteetin hyödyntämistä.

Fototriangulação em bloco de imagens orbitais com modelos rigorosos baseados em pontos e retas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Retificador trifásico de 18 pulsos com estágio CC controlado por histerese constante

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Serial bone marrow transplantation reveals in vivo expression of the pCLPG retroviral vector

Abstract Background Gene therapy in the hematopoietic system remains promising, though certain aspects of vector design, such as transcriptional control elements, continue to be studied. Our group has developed a retroviral vector where transgene expression is controlled by p53 with the intention of harnessing the dynamic and inducible nature of this tumor suppressor and transcription factor. We present here a test of in vivo expression provided by the p53-responsive vector, pCLPG. For this, we used a model of serial transplantation of transduced bone marrow cells. Results We observed, by flow cytometry, that the eGFP transgene was expressed at higher levels when the pCLPG vector was used as compared to the parental pCL retrovirus, where expression is directed by the native MoMLV LTR. Expression from the pCLPG vector was longer lasting, but did decay along with each sequential transplant. The detection of eGFP-positive cells containing either vector was successful only in the bone marrow compartment and was not observed in peripheral blood, spleen or thymus. Conclusions These findings indicate that the p53-responsive pCLPG retrovirus did offer expression in vivo and at a level that surpassed the non-modified, parental pCL vector. Our results indicate that the pCLPG platform may provide some advantages when applied in the hematopoietic system.

Inducible endothelial cell-specific gene expression in transgenic mouse embryos and adult mice

Utilizing both the TET-OFF and TET-ON systems in combination with transcriptional control elements of the Tie-2 gene, we have established a series of transgenic activator and responder mice for TET-regulated endothelial cell-specific transgene expression in double transgenic mouse embryos and in adult mice. TET-regulated expression of LacZ reporter genes could be achieved in virtually all endothelia in mid gestation stage mouse embryos. In contrast in adult mice, using the very same Tie-2 tTA activator mouse strain, we observed striking differences of TET-induced gene expression from various inducible expression constructs in different vascular beds. Non-endothelial expression was never detected. The prominent differences in completeness of TET-induced endothelial expression highlight the still underestimated critical role of the responder mouse lines for uniform TET-induced gene expression in heterogeneous cell populations such as endothelial cells. Interestingly, in double transgenic mice inducibly expressing several different adhesion molecules, no adverse effects were observed even though these proteins were robustly expressed on endothelial cells in adult tissues. These transgenic model systems provide versatile tools for the TET-regulated manipulation of endothelial cell-specific gene expression in the entire embryonic vasculature and distinct vascular beds in adult mice.

Sistema de autenticación accesible para el hogar digital

El Hogar Digital Accesible (HDA) de la ETSIST nace con el propósito de acercar las nuevas Tecnologías de la Información a las personas que precisan de necesidades concretas de accesibilidad y usabilidad, dotándoles de herramientas que les permitan aumentar su calidad de vida, confort, seguridad y autonomía. El entorno del HDA consta de elementos de control para puertas, persianas, iluminación, agua o gas, sensores de temperatura, incendios, gas, sistemas de climatización, sistemas de entretenimiento y sistemas de seguridad tales como detectores de presencia y alarmas. Todo ello apoyado sobre una arquitectura de red que proporciona una pasarela residencial y un acceso a banda ancha. El objetivo principal de este PFG ha sido el desarrollo de un sistema de autenticación para el Hogar Digital Accesible de bajo coste. La idea de integrar un sistema de autenticación en el HDA, surge de la necesidad de proteger de accesos no deseados determinados servicios disponibles dentro de un ámbito privado. Algunos de estos servicios pueden ser tales como el acceso a la lectura de los mensajes disponibles en el contestador automático, el uso de equipos multimedia, la desconexión de alarmas de seguridad o simplemente la configuración de ambientes según el usuario que esté autenticado (intensidad de luz, temperatura de la sala, etc.). En el desarrollo han primado los principios de accesibilidad, usabilidad y seguridad necesarios para la creación de un entorno no invasivo, que permitiera acreditar la identidad del usuario frente al sistema HDA. Se ha planteado como posible solución, un sistema basado en el reconocimiento de un trazo realizado por el usuario. Este trazo se usará como clave de cara a validar a los usuarios. El usuario deberá repetir el trazado que registró en el sistema para autenticarse. Durante la ejecución del presente PFG, se justificará la elección de este mecanismo de autenticación frente a otras alternativas disponibles en el mercado. Para probar la aplicación, se ha podido contar con dos periféricos de distintas gamas, el uDraw creado para la PS3 que se compone de una tableta digitalizadora y un lápiz que permite recoger los trazos realizados por el usuario de forma inalámbrica y la tableta digitalizadora Bamboo de Wacom. La herramienta desarrollada permite a su vez, la posibilidad de ser usada por otro tipo de dispositivos como es el caso del reloj con acelerómetro de 3 ejes de Texas Instruments Chronos eZ430 capaz de trasladar los movimientos del usuario al puntero de un ratón. El PFG se encuentra dividido en tres grandes bloques de flujo de trabajo. El primero se centra en el análisis del sistema y las tecnologías que lo componen, incluyendo los distintos algoritmos disponibles para realizar la autenticación basada en reconocimiento de patrones aplicados a imágenes que mejor se adaptan a las necesidades del usuario. En el segundo bloque se recoge una versión de prueba basada en el análisis y el diseño UML realizado previamente, sobre la que se efectuaron pruebas de concepto y se comprobó la viabilidad del proyecto. El último bloque incluye la verificación y validación del sistema mediante pruebas que certifican que se han alcanzado los niveles de calidad necesarios para la consecución de los objetivos planteados, generando finalmente la documentación necesaria. Como resultado del trabajo realizado, se ha obtenido un sistema que plantea una arquitectura fácilmente ampliable lograda a través del uso de técnicas como la introspección, que permiten separar la lógica de la capa de negocio del código que la implementa, pudiendo de forma simple e intuitiva sustituir código mediante ficheros de configuración, lo que hace que el sistema sea flexible y escalable. Tras la realización del PFG, se puede concluir que el producto final obtenido ha respondido de forma satisfactoria alcanzando los niveles de calidad requeridos, siendo capaz de proporcionar un sistema de autenticación alternativo a los convencionales, manteniendo unas cotas de seguridad elevadas y haciendo de la accesibilidad y el precio sus características más reseñables. ABSTRACT. Accessible Digital Home (HDA) of the ETSIST was created with the aim of bringing the latest information and communications technologies closer to the people who has special needs of accessibility and usability increasing their quality of life, comfort, security and autonomy. The HDA environment has different control elements for doors, blinds, lighting, water or gas, temperature sensors, fire protection systems, gas flashover, air conditioning systems, entertainments systems and security systems such as intruders detectors and alarms. Everything supported by an architecture net which provides a broadband residential services gateway. The main goal of this PFG was the development of a low-cost authentication system for the Accessible Digital Home. The idea of integrating an authentication system on the HDA, stems from the need to safeguard certain private key network resources from unauthorized access. Some of said resources are the access to the answering machine messages, the use of multimedia devices, the alarms deactivation or the parameter settings for each environment as programmed by the authenticated user (light intensity, room temperature, etc.). During the development priority was given to concepts like accessibility, usability and security. All of them necessary to create a non invasive environment that allows the users to certify their identity. A system based on stroke pattern recognition, was considered as a possible solution. This stroke is used as a key to validate users. The user must repeat the stroke that was saved on the system to validate access. The selection of this authentication mechanism among the others available options will be justified during this PFG. Two peripherals with different ranges were used to test the application. One of them was uDraw design for the PS3. It is wireless and is formed by a pen and a drawing tablet that allow us to register the different strokes drawn by the user. The other one was the Wacom Bamboo tablet, that supports the same functionality but with better accuracy. The developed tool allows another kind of peripherals like the 3-axes accelerometer digital wristwatch Texas Instruments Chronos eZ430 capable of transfering user movements to the mouse cursor. The PFG is divided by three big blocks that represent different workflows. The first block is focused on the system analysis and the technologies related to it, including algorithms for image pattern recognition that fits the user's needs. The second block describes how the beta version was developed based on the UML analysis and design previously done. It was tested and the viability of the project was verified. The last block contains the system verification and validation. These processes certify that the requirements have been fulfilled as well as the quality levels needed to reach the planned goals. Finally all the documentation has been produced. As a result of the work, an expandable system has been created, due to the introspection that provides the opportunity to separate the business logic from the code that implements it. With this technique, the code could be replaced throughout configuration files which makes the system flexible and highly scalable. Once the PFG has finished, it must therefore be concluded that the final product has been a success and high levels of quality have been achieved. This authentication tool gives us a low-cost alternative to the conventional ones. The new authentication system remains security levels reasonably high giving particular emphasis to the accessibility and the price.

Engineering precision RNA molecular switches

Ligand-specific molecular switches composed of RNA were created by coupling preexisting catalytic and receptor domains via structural bridges. Binding of ligand to the receptor triggers a conformational change within the bridge, and this structural reorganization dictates the activity of the adjoining ribozyme. The modular nature of these tripartite constructs makes possible the rapid construction of precision RNA molecular switches that trigger only in the presence of their corresponding ligand. By using similar enzyme engineering strategies, new RNA switches can be made to operate as designer molecular sensors or as a new class of genetic control elements.

A transgenic mouse model to analyze CD8+ effector T cell differentiation in vivo

Antigen-specific effector T cells are prerequisite to immune protection, but because of the lack of effector cell-specific markers, their generation and differentiation has been difficult to study. We report that effector cells are highly enriched in a T cell subset that can be specifically identified in transgenic (T-GFP) mice expressing green fluorescent protein (GFP) under control of the murine CD4 promoter and proximal enhancer. Consistent with previous studies of these transcriptional control elements, GFP was strongly and specifically expressed in nearly all resting and short-term activated CD4+ and CD8+ T cells. However, when T-GFP mice were challenged with vaccinia virus, allogeneic tumor cells, or staphylococcal enterotoxin A, the cytotoxic and IFN-γ-producing T cells lost GFP expression. Upon T cell receptor (TCR) ligation by αCD3, sorted GFP+ cells fluxed calcium and proliferated vigorously. In contrast, GFP− effector cells showed a diminished calcium flux and did not proliferate. Instead, they underwent apoptosis unless supplied with exogenous IL-2. By reverse transcription–PCR analysis, the GFP− cells up-regulated the pro-apoptotic molecule, Fas-L, and down-regulated gene expression of the proximal TCR signaling molecule, CD3ζ, and c-jun, a component of the AP-1 transcription factor. Thus, differential regulation of TCR signaling may explain the divergent responses of naïve and effector T cells to antigen stimulation.

Efficient studies of long-distance Bmp5 gene regulation using bacterial artificial chromosomes

The regulatory regions surrounding many genes may be large and difficult to study using standard transgenic approaches. Here we describe the use of bacterial artificial chromosome clones to rapidly survey hundreds of kilobases of DNA for potential regulatory sequences surrounding the mouse bone morphogenetic protein-5 (Bmp5) gene. Simple coinjection of large insert clones with lacZ reporter constructs recapitulates all of the sites of expression observed previously with numerous small constructs covering a large, complex regulatory region. The coinjection approach has made it possible to rapidly survey other regions of the Bmp5 gene for potential control elements, to confirm the location of several elements predicted from previous expression studies using regulatory mutations at the Bmp5 locus, to test whether Bmp5 control regions act similarly on endogenous and foreign promoters, and to show that Bmp5 control elements are capable of rescuing phenotypic effects of a Bmp5 deficiency. This rapid approach has identified new Bmp5 control regions responsible for controlling the development of specific anatomical structures in the vertebrate skeleton. A similar approach may be useful for studying complex control regions surrounding many other genes important in embryonic development and human disease.

CDP/cut is the DNA-binding subunit of histone gene transcription factor HiNF-D: a mechanism for gene regulation at the G1/S phase cell cycle transition point independent of transcription factor E2F.

Transcription of the genes for the human histone proteins H4, H3, H2A, H2B, and H1 is activated at the G1/S phase transition of the cell cycle. We have previously shown that the promoter complex HiNF-D, which interacts with cell cycle control elements in multiple histone genes, contains the key cell cycle factors cyclin A, CDC2, and a retinoblastoma (pRB) protein-related protein. However, an intrinsic DNA-binding subunit for HiNF-D was not identified. Many genes that are up-regulated at the G1/S phase boundary are controlled by E2F, a transcription factor that associates with cyclin-, cyclin-dependent kinase-, and pRB-related proteins. Using gel-shift immunoassays, DNase I protection, and oligonucleotide competition analyses, we show that the homeodomain protein CDP/cut, not E2F, is the DNA-binding subunit of the HiNF-D complex. The HiNF-D (CDP/cut) complex with the H4 promoter is immunoreactive with antibodies against CDP/cut and pRB but not p107, whereas the CDP/cut complex with a nonhistone promoter (gp91-phox) reacts only with CDP and p107 antibodies. Thus, CDP/cut complexes at different gene promoters can associate with distinct pRB-related proteins. Transient coexpression assays show that CDP/cut modulates H4 promoter activity via the HiNF-D-binding site. Hence, DNA replication-dependent histone H4 genes are regulated by an E2F-independent mechanism involving a complex of CDP/cut with cyclin A/CDC2/ RB-related proteins.

Hepatitis B virus transactivator protein, HBx, associates with the components of TFIIH and stimulates the DNA helicase activity of TFIIH.

Human hepatitis B virus genome encodes a protein, termed HBx, that is widely recognized as a transcriptional transactivator. While HBx does not directly bind cis-acting transcriptional control elements, it has been shown to associate with cellular proteins that bind DNA. Because HBx transactivated a large number of viral/cellular transcriptional control elements, we looked for its targets within the components of the basal transcriptional machinery. This search led to the identification of its interactions with TFIIH. Here, we show that HBx interacts with yeast and mammalian TFIIH complexes both in vitro and in vivo. These interactions between HBx and the components of TFIIH are supported by several lines of evidence including results from immunoprocedures and direct methods of measuring interactions. We have identified ERCC3 and ERCC2 DNA helicase subunits of holoenzyme TFIIH as targets of HBx interactions. Furthermore, the DNA helicase activity of purified TFIIH from rat liver and, individually, the ERCC2 component of TFIIH is stimulated in the presence of HBx. These observations suggest a role for HBx in transcription and DNA repair.

Characterization of promoters and stable transfection by homologous and nonhomologous recombination in Plasmodium falciparum.

Genetic studies of the protozoan parasite Plasmodium falciparum have been severely limited by the inability to introduce or modify genes. In this paper we describe a system of stable transfection of P. falciparum using a Toxoplasma gondii dihydrofolate reductase-thymidylate synthase gene, modified to confer resistance to pyrimethamine, as a selectable marker. This gene was placed under the transcriptional control of the P. falciparum calmodulin gene flanking sequences. Transfected parasites generally maintained plasmids episomally while under selection; however, parasite clones containing integrated forms of the plasmid were obtained. Integration occurred by both homologous and nonhomologous recombination. In addition to the flanking sequence of the P. falciparum calmodulin gene, the 5' sequences of the P. falciparum and P. chabaudi dihydrofolate reductase-thymidylate synthase genes were also shown to be transcriptionally active in P. falciparum. The minimal 5' sequence that possessed significant transcriptional activity was determined for each gene and short sequences containing important transcriptional control elements were identified. These sequences will provide considerable flexibility in the future construction of plasmid vectors to be used for the expression of foreign genes or for the deletion or modification of P. falciparum genes of interest.

Transcription of the lymphocyte-specific terminal deoxynucleotidyltransferase gene requires a specific core promoter structure.

The terminal deoxynucleotidyltransferase (TdT) gene encodes a template-independent DNA polymerase that is expressed exclusively in immature lymphocytes. The TdT promoter lacks a TATA box, but an initiator element (Inr) overlaps the transcription start site. The Inr directs basal transcription and also mediates activated transcription in conjunction with an upstream element called D'. We have begun to address the fundamental question of why the TdT promoter contains an Inr rather than a TATA box. First, we tested the possibility that the TdT promoter lacks a TATA box because the -30 region is needed for the binding of an essential regulator. Mutations were introduced into the -30 region, and the mutants were tested in transient transfection and in vitro transcription assays. The mutations had only minor effects on promoter strength, suggesting that this first hypothesis is incorrect. Next, the effect of inserting a TATA box within the -30 region was tested. Although the TATA box enhanced promoter strength, appropriate regulation appeared to be maintained, as transcription in lymphocytes remained dependent on the D' element. Finally, a promoter variant containing a TATA box at -30, but a mutant Inr, was tested. Surprisingly, transcription from this variant, both in vitro and in vivo, was dramatically reduced. These results suggest that the TdT promoter, and possibly other natural promoters, contain an Inr element because one or more activator proteins that interact with surrounding control elements preferentially function in its presence.

Expression of the human acute myeloid leukemia gene AML1 is regulated by two promoter regions.

The human chromosome 21 AML1 gene is expressed predominantly in the hematopoietic system. In several leukemia-associated translocations AML1 is fused to other genes and transcription of the fused regions is mediated by upstream sequences that normally regulate the expression of AML1. The 5' genomic region of AML1 was cloned and sequenced. The two 5' untranslated regions (UTRs) previously identified in AML1 cDNAs were located in this region and the distance between them was established. The distal 5' UTR maps over 7 kb upstream of the proximal one. Using primer extension with mRNA, transcription start sites were identified at two distinct sites above these 5' uTRs. Sequence analysis revealed the absence of a TATA motif and the presence of Sp1, PU.1, Oct, CRE, Myb, Ets, and Ets-like binding sites in both upstream regions. Several initiator elements (Inr) that overlap the transcription start sites were also identified. These proximal and distal upstream regions and their deletion mutants were cloned in front of a luciferase reporter gene and used in transfection assays. We demonstrate that both upstream regions function as promoters in hematopoietic (Jurkat) and nonhematopoietic (HEK) cell lines. The activity of both promoters was orientation dependent and was enhanced, in a cell-type specific manner, by a heterologous enhancer sequence. These results indicate that additional control elements, either negative or positive, regulate the tissue-specific expression of AML1.

Functional interactions between the retinoblastoma (Rb) protein and Sp-family members: superactivation by Rb requires amino acids necessary for growth suppression.

The transient expression of the retinoblastoma protein (Rb) regulates the transcription of a variety of growth-control genes, including c-fos, c-myc, and the gene for transforming growth factor beta 1 via discrete promoter sequences termed retinoblastoma control elements (RCE). Previous analyses have shown that Sp1 is one of three RCE-binding proteins identified in nuclear extracts and that Rb functionally interacts with Sp1 in vivo, resulting in the "superactivation" of Sp1-mediated transcription. By immunochemical and biochemical criteria, we report that an Sp1-related transcription factor, Sp3, is a second RCE-binding protein. Furthermore, in transient cotransfection assays, we report that Rb "superactivates" Sp3-mediated RCE-dependent transcription in vivo and that levels of superactivation are dependent on the trans-activator (Sp1 or Sp3) studied. Using expression vectors carrying mutated Rb cDNAs, we have identified two portions of Rb required for superactivation: (i) a portion of the Rb "pocket" (amino acids 614-839) previously determined to be required for physical interactions between Rb and transcription factors such as E2F-1 and (ii) a novel amino-terminal region (amino acids 140-202). Since both of these regions of Rb are targets of mutation in human tumors, our data suggest that superactivation of Sp1/Sp3 may play a role in Rb-mediated growth suppression and/or the induction of differentiation.