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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
121

La disolución parlamentaria en el Perú

Cairo Roldán, Omar 25 September 2017 (has links)
En este artículo se busca esclarecer el significado de la institución de la disolución parlamentaria. En primer término, se revisan sus orígenes y los usos que se dio a esta institución en Inglaterra. En segundo lugar, se explica que la disolución parlamentaria es una institución perteneciente al ordenamiento constitucional de un país, y que sirve para viabilizar su funcionamiento pero no para destruir ese ordenamiento mediante un golpe de Estado. Finalmente, se examina cómo fue regulada en el Perú la disolución parlamentaria en la Constitución de 1979, y cómo se encuentra regulada actualmente en la Constitución de 1993 vigente en nuestro país.
122

A influência da água na composição de primers autocondicionantes experimentais / Influence of the water concentration in an experimental self-etching primer on the bond strength to dentin

Lima, Giana da Silveira 21 December 2007 (has links)
Made available in DSpace on 2014-08-20T14:30:07Z (GMT). No. of bitstreams: 1 Dissertacao_giana_da_silveira_lima.pdf: 1493174 bytes, checksum: 68e4c67538ad95eba022c0a0e790d4b8 (MD5) Previous issue date: 2007-12-21 / To investigate the influence of different water concentrations in self-etching primers solvents on microtensile bond strength (μTBS) of an experimental adhesive system. Five experimental self-etching primers with 0, 5, 10, 20 and 40 water concentration (wt %) in solvent, were synthesized. Clearfil SE Bond (CSEB) was used as commercial adhesive. Sixty bovine incisive were randomize separated in six groups (n=20). Vestibular enamel was removed to expose the superficial coronal dentin, this surface was polished wet to create a standardized smear layer. After rinsing, water was removed, leaving the surface visibly dried. The dentin surfaces were etched with primer and air-dried, after coating adhesive resin was applied and photo-activated, following the composite resin restoration was accomplished. After storage for 24 hours, the specimens were sectioned with a refrigerated diamond saw at low-speed. Microtensile bond strength were measured and data were analyzed by one-way ANOVA/Tukey s test (α=0.05). Analysis of variance showed that primer composition was a significant factor for bond strength. There was not difference on bond strength of the primers with water concentration: 40% (53.9 ± 12.7 MPa), 20% (51.1 ± 11.5 MPa) and 10% (47.5 ± 11.4 MPa) and CSEB (52.44 ± 13.27 MPa). The group with 5% (38.6 ± 12.9 MPa) and 0% (31.5 ± 7.5 MPa), present similar bond strength amongst themselves but statistically smaller that the other groups. The water concentration, present in the primer solvent, exercise significant influence on the bond strength of this experimental self-etching adhesive system / Este estudo avaliou a influência de diferentes concentrações de água no solvente de um primer autocondicionante de um sistema adesivo dentinário experimental. Foram formulados cinco primers experimentais com diferentes concentrações, 0; 5; 10; 20; 40%, de água em massa e Clearfil SE Bond (CSEB) foi utilizado como referência comercial. Foram utilizados 60 incisivos bovinos, distribuídos aleatoriamente entre 6 grupos (n= 20). Na face vestibular dos dentes foi realizado desgaste com lixas até a exposição de dentina com lixa d água 600. O primer foi aplicado sobre a dentina previamente seca com papel absorvente, sendo aplicado jato de ar e em seguida o adesivo foi aplicado e foto-ativado com aparelho fotopolimerizador LED SDI Radii®. A restauração foi confeccionada utilizando 2 incrementos, fotoativados de acordo com as recomendações do fabricante. Após armazenagem por 24 horas em água destilada à 37°C, os dentes foram seccionados em cortadeira de precisão e a resistência de união mensurada, através de ensaio de microtração em uma máquina de ensaios mecânicos. Análise de Variância segundo um critério e teste complementar de Tukey foram utilizados na análise estatística (α=5%). Não houve diferença na resistência de união de P40 (53,9 ±12,7 MPa) P20 (51,1 ±11,5 MPa) e P10 (47,5 ±11,4 MPa) e CB (52,44 ±13,27 MPa). Os grupos P5 (38,6 ±12,9 MPa) e P0 (31.5 ±7.5 MPa) não apresentaram diferença estatística entre si, mas seus valores de adesão dentinária foram menores que os demais grupos (p<0,05). A resistência de união dentinária do sistema adesivo experimental testado sofre influência da concentração de água presente no solvente do primer autocondicionante
123

Analysis of singularities in elliptic equations : the Ginzburg-Landau model of superconductivity, the Lin-Ni-Takagi problem, the Keller-Segel model of chemotaxis, and conformal geometry / Analyse des singularités dans les équations elliptiques : le modèle de superconductivité Ginzburg-Landau, le problème Lin-Ni-Takagi, le modèle Keller-Segel de chimiotaxie , et la géométrie conforme

Román, Carlos 15 December 2017 (has links)
Cette thèse est consacrée à l'analyse des singularités apparaissant dans des équations différentielles partielles elliptiques non linéaires découlant de la physique mathématique, de la biologie mathématique, et de la géométrie conforme. Les thèmes abordés sont le modèle de supraconductivité de Ginzburg-Landau, le problème de Lin-Ni-Takagi, le modèle de Keller-Segel de la chimiotaxie, et le problème de courbure scalaire prescrite. Le modèle de Ginzburg-Landau est une description phénoménologique de la supraconductivité. Une caractéristique essentielle des supraconducteurs de type II est la présence de vortex, qui apparaissent au-dessus d'une certaine valeur de la force du champ magnétique appliqué, appelée premier champ critique. Nous nous intéressons au régime de epsilon petit, où epsilon est l'inverse du paramètre de Ginzburg-Landau (une constante du matériau). Dans ce régime, les vortex sont au premier ordre des singularités topologiques de co-dimension 2. Nous fournissons une construction quantitative par approximation de vortex en dimension trois pour l'énergie de Ginzburg-Landau, ce qui donne une approximation des lignes de vortex ainsi qu'une borne inférieure pour l'énergie, qui est optimale au premier ordre et vérifiée au niveau epsilon. En utilisant ces outils, nous analysons ensuite le comportement des minimiseurs globaux en dessous et proche du premier champ critique. Nous montrons que, en dessous de cette valeur critique, les minimiseurs de l'énergie de Ginzburg-Landau sont des configurations sans vortex et que les minimiseurs, proche de cette valeur, ont une vorticité bornée. Le problème de Lin-Ni-Takagi apparait comme l'ombre (dans la littérature anglaise ``shadow'') du système de Gierer-Meinhardt d'équations de réaction-diffusion qui modélise la formation de motifs biologiques. Ce problème est celui de trouver des solutions positives d'une équation critique dans un domaine régulier et borné de dimension trois, avec une condition de Neumann homogène au bord. Dans cette thèse, nous construisons des solutions à ce problème présentant un comportement explosif en un point du domaine, lorsqu'un certain paramètre converge vers une valeur critique. La chimiotaxie est l'influence de substances chimiques dans un environnement sur le mouvement des organismes. Le modèle de Keller-Segel pour la chimiotaxie est un système de diffusion-advection composé de deux équations paraboliques couplées. Ici, nous nous intéressons aux états stationnaires radiaux de ce système. Nous sommes alors amenés à étudier une équation critique dans la boule unité de dimension 2, avec une condition de Neumann homogène au bord. Dans cette thèse, nous construisons plusieurs familles de solutions radiales qui explosent à l'origine de la boule, et se concentrent sur le bord et/ou sur une sphère intérieure, lorsqu' un certain paramètre converge vers zéro. Enfin, nous étudions le problème de la courbure scalaire prescrite. Étant donnée une variété Riemannienne compacte de dimension n, nous voulons trouver des métriques conformes dont la courbure scalaire soit une fonction prescrite, qui dépend d'un petit paramètre. Nous supposons que cette fonction a un point critique qui satisfait une hypothèse de platitude appropriée. Nous construisons plusieurs métriques, qui explosent lorsque le paramètre converge vers zéro, avec courbure scalaire prescrite. / This thesis is devoted to the analysis of singularities in nonlinear elliptic partial differential equations arising in mathematical physics, mathematical biology, and conformal geometry. The topics treated are the Ginzburg-Landau model of superconductivity, the Lin-Ni-Takagi problem, the Keller-Segel model of chemotaxis, and the prescribed scalar curvature problem. The Ginzburg-Landau model is a phenomenological description of superconductivity. An essential feature of type-II superconductors is the presence of vortices, which appear above a certain value of the strength of the applied magnetic field called the first critical field. We are interested in the regime of small epsilon, where epsilon is the inverse of the Ginzburg-Landau parameter (a material constant). In this regime, the vortices are at main order co-dimension 2 topological singularities. We provide a quantitative three-dimensional vortex approximation construction for the Ginzburg-Landau energy, which gives an approximation of vortex lines coupled to a lower bound for the energy, which is optimal to leading order and valid at the epsilon-level. By using these tools we then analyze the behavior of global minimizers below and near the first critical field. We show that below this critical value, minimizers of the Ginzburg-Landau energy are vortex-free configurations and that near this value, minimizers have bounded vorticity. The Lin-Ni-Takagi problem arises as the shadow of the Gierer-Meinhardt system of reaction-diffusion equations that models biological pattern formation. This problem is that of finding positive solutions of a critical equation in a bounded smooth three-dimensional domain, under zero Neumann boundary conditions. In this thesis, we construct solutions to this problem exhibiting single bubbling behavior at one point of the domain, as a certain parameter converges to a critical value. Chemotaxis is the influence of chemical substances in an environment on the movement of organisms. The Keller-Segel model for chemotaxis is an advection-diffusion system consisting of two coupled parabolic equations. Here, we are interested in radial steady states of this system. We are then led to study a critical equation in the two-dimensional unit ball, under zero Neumann boundary conditions. In this thesis, we construct several families of radial solutions which blow up at the origin of the ball and concentrate on the boundary and/or an interior sphere, as a certain parameter converges to zero. Finally, we study the prescribed scalar curvature problem. Given an n-dimensional compact Riemannian manifold, we are interested in finding bubbling metrics whose scalar curvature is a prescribed function, depending on a small parameter. We assume that this function has a critical point which satisfies a suitable flatness assumption. We construct several metrics, which blow-up as the parameter goes to zero, with prescribed scalar curvature.
124

Návrh a konstrukce dvoukolového mobilního robotu / Design and Construction of a Two-Wheel Mobile Robot

Meisl, Milan January 2011 (has links)
This diploma thesis deals with a design and construction of a two-wheeled mobile robot. In order to prepare a high-quality project, a testing carriage has been constructed serving as a basis of useful information for the final design and construction. Beside introduction and conclusion, the diploma thesis consists of four main parts. While the first part of the thesis briefly introduces the field of robotics, the theoretical part focuses both on particular components necessary for the robot's construction as well as the Segway vehicle which served as an inspiration for the robot's functionality. After characteristics of a testing carriage, attention is devoted to individual methods of stabilization, with several different types of sensors being used. Furthermore, the chapter on mechanical design examines choice of devices and their position and provides a scheme for their construction. The electrical design follows afterwards, describing circuits that were considered optimal for the designed wiring. The forth part of the diploma thesis concentrates on the construction of a two-wheeled carriage and covers following topics: construction of mechanical components, production of electric boards, programmatic equipment of the carriage and its implementation, testing of the carriage and finally also an evaluation of achieved results.
125

Développement d’un primaire d’adhésion anticorrosion sans Cr VI pour l’alliage aéronautique Al2024-T3 / Development of an anticorrosive primer without hexavalent chromium for AA2024-T3

Doublet, Aurélien 26 September 2019 (has links)
L’utilisation du chrome hexavalent pour le traitement de surface des matériaux de l’aéronautique est en passe d’être interdite par la réglementation REACH. Dans l’objectif de trouver une solution alternative, PROTEC industrie et le CEA travaillent en collaboration sur le développement d’une nouvelle solution sans-chrome permettant de remplacer les revêtements classiquement utilisés. Le travail de thèse présenté dans ce manuscrit s’inscrit dans le cadre du laboratoire commun MESTREL financé par le programme ANR Labcom, réunissant les deux entités citées ci-dessus. La solution proposée est basée sur le greffage d’un revêtement organique inspiré de la chimie des sels de diazoniums à la surface de l’Al2024-T3, alliage de référence du secteur aéronautique. Cette thèse vise initialement à déterminer les conditions pour lesquelles les propriétés du film greffé se rapprochent le plus de celles de son prédécesseur. Pour cela, des caractérisations spectroscopiques et électrochimiques des films greffés ont été réalisées. Les protocoles de microscopies électrochimiques développés ont notamment permis de sonder le niveau de passivation de la couche. La haute porosité du film greffé, nous a ainsi permis d’imaginer et de développer un système bicouche innovant combinant les propriétés anticorrosion des solutions actuelles aux hautes propriétés d’adhérence du film organique fonctionnalisé. En parallèle, une méthode de dépôt hors-cuve a été développée pour générer un gain économique important et faciliter le traitement de pièces de grandes dimensions. / The use of hexavalent chromium for anticorrosion surface treatments in aeronautics field will soon be banned by the REACH reglementation. Looking for an innovative solution, PROTEC industry and the CEA research center work in collaboration with the aim of developing a chromium free replacement treatment, which can substitute current solution and give competitive results both in adhesion and corrosion protection. The thesis project presented in this manuscript is part of a common laboratory named MESTREL, including the two entities. The proposed solution is based on the grafting of a polymeric film inspired by the diazonium chemistry on an Al2024-T3 surface, reference alloy in the aeronautic field. The principal objective of the thesis is to determinate the best conditions of grafting, -giving similar properties to chromium treatment. To this end, spectroscopic and electrochemical characterizations have been performed. Electrochemical Microscopy protocols are developed to assess the passivation properties of the film The high porosity of the film has allowed to develop a new innovative bilayer process combining anticorrosive properties of current solution and adhesion properties of the functionnal coating. At the same time, an out-of-tank deposition technique is developed. The goal is to enable economic benefits and facilitate treatment of very large aeronautic pieces.
126

Radical critique and eschatology : the chronicle of a sixteenth-century Peruvian Indian

Nash, Mark G. (Mark Guy) January 1993 (has links)
No description available.
127

Thermodynamics of λ-PCR Primer Design and Effective Ribosome Binding Sites

Berg, Emily Katherine 07 June 2019 (has links)
Recombinant DNA technology has been commonly used in a number of fields to synthesize new products or generate products with a new pathway. Conventional cloning methods are expensive and require significant time and labor; λ-PCR, a new cloning method developed in the Senger lab, has a number of advantages compared to other cloning processes due to its employment of relatively inexpensive and widely available materials and time-efficiency. While the amount of lab work required for the cloning process is minimal, the importance of accurate primer design cannot be overstated. The target of this study was to create an effective procedure for λ-PCR primer design that ensures accurate cloning reactions. Additionally, synthetic ribosome binding sites (RBS) were included in the primer designs to test heterologous protein expression of the cyan fluorescent reporter with different RBS strengths. These RBS sequences were designed with an online tool, the RBS Calculator. A chimeric primer design procedure for λ-PCR was developed and shown to effectively create primers used for accurate cloning with λ-PCR; this method was used to design primers for CFP cloning in addition to two enzymes cloned in the Senger lab. A total of five strains of BL21(DE3) with pET28a + CFP were constructed, each with the same cyan fluorescent protein (CFP) reporter but different RBS sequences located directly upstream of the start codon of the CFP gene. Expression of the protein was measured using both whole-cell and cell-free systems to determine which system yields higher protein concentrations. A number of other factors were tested to optimize conditions for high protein expression, including: induction time, IPTG concentration, temperature, and media (for the cell-free experiments only). Additionally, expression for each synthetic RBS sequence was investigated to determine an accurate method for predicting protein translation. NUPACK and the Salis Lab RBS Calculator were both used to evaluate the effects of these different synthetic RBS sequences. The results of the plate reader experiments with the 5 CFP strains revealed a number of factors to be statistically significant when predicting protein expression, including: IPTG concentration, induction time, and in the cell-free experiments, type of media. The whole-cell system consistently produced higher amounts of protein than the cell-free system. Lastly, contrasts between the CFP strains showed each strain's performance did not match the predictions from the RBS Calculator. Consequently, a new method for improving protein expression with synthetic RBS sequences was developed using relationships between Gibbs free energy of the RBS-rRNA complex and expression levels obtained through experimentation. Additionally, secondary structure present at the RBS in the mRNA transcript was modeled with strain expression since these structures cause deviations in the relationship between Gibbs free energy of the mRNA-rRNA complex and CFP expression. / Master of Science / Recombinant DNA technology has been used to genetically enhance organisms to produce greater amounts of a product already made by the organism or to make an organism synthesize a new product. Genes are commonly modified in organisms using cloning practices which typically involves inserting a target gene into a plasmid and transforming the plasmid into the organism of interest. A new cloning process developed in the Senger lab, λ-PCR, improves the cloning process compared to other methods due to its use of relatively inexpensive materials and high efficiency. A primary goal of this study was to develop a procedure for λ-PCR primer design that allows for accurate use of the cloning method. Additionally, this study investigated the use of synthetic ribosome binding sites to control and improve expression of proteins cloned into an organism. Ribosome binding sites are sequences located upstream of the gene that increase the molecule’s affinity for the rRNA sequence on the ribosome, bind to the ribosome just upstream of the beginning of the gene, and initiate expression of the gene. Tools have been developed that create synthetic ribosome binding sites designed to produce specific amounts of protein. For example, the tools can increase or decrease expression of a gene depending on the application. These tools, the Salis Lab RBS Calculator and NUPACK, were used to design and evaluate the effects of the synthetic ribosome binding sites. Additionally, a new method was created to design synthetic ribosome binding sites since the methods used during the design process yielded inaccuracies. Each strain of E. coli contained the same gene, a cyan fluorescent protein (CFP), but had different RBS sequences located upstream of the gene. Expression of CFP was controlled via induction, meaning the addition of a particular molecule, IPTG in this system, triggered expression of CFP. Each of the CFP strains were tested with a variety of v conditions in order to find the conditions most suitable for protein expression; the variables tested include: induction time, IPTG (inducer) concentration, and temperature. Media was also tested for the cell-free systems, meaning the strains were grown overnight for 18 hours and lysed, a process where the cell membrane is broken in order to utilize the cell’s components for protein expression; the cell lysate was resuspended in new media for the experiments. ANOVA and multiple linear regression revealed IPTG concentration, induction time, and media to be significant factors impacting protein expression. This analysis also showed each CFP strain did not perform as the RBS Calculator predicted. Modeling each strain’s CFP expression using the RBS-rRNA binding strengths and secondary structures present in the RBS allowed for the creation of a new model for predicting and designing RBS sequences.
128

Caractérisation et ciblage de la reconnaissance dynamique de Trp37-G lors de l’interaction de la protéine NCp7 de HIV-1 avec des acides nucléiques / Characterization and targeting the dynamic recognition of Trp37-G during the interaction of NCp7 protein of HIV-1 with nucleic acids

Sharma, Rajhans 10 April 2018 (has links)
La protéine de la nucléocapside (NC) possède un rôle important dans le cycle de viral du VIH-1 grâce à sa propriété chaperone des acides nucléiques (NA) qui implique la reconnaissance de son résidu Trp37 avec un résidu Guanine de l'acide nucléique cible. Nous avons caractérisé cette reconnaissance dynamique Trp37-G en utilisant des séquences impliquées dans la transcription inverse et l'assemblage de l'ARN génomique. En utilisant les analogues nucléosidiques fluorescents thienoguanosine (thG) et 2-thiényl-3-hydroxychromone (3HCnt), nous avons déterminé l'ensemble des constantes de vitesse cinétiques du mécanisme d’hybridation de la séquence (-)PBS avec (+)PBS en absence et en présence de NC. Nous avons également étudié le rôle du NA sucre dans les complexes NC-ARN et NC-ADN, puisque la protéine NC se lie avec la polarité opposée aux séquences d'ADN et d'ARN. Nous avons confirmé que l'interaction du résidu Trp37 avec les amino-acides de type guanines était critique lors de la formation des complexes avec les deux mutants d’ARN et d’ADN de PBS et de SL3. Enfin, nous avons réalisé un criblage de potentiels inhibiteurs de la protéine NC et examiné les touches identifiées à partir d’un test basé sur la fluorescence de la sonde thG. / Nucleocapsid protein (NC) plays crucial roles in HIV-1 life cycle through its nucleic acid (NA) chaperoning property that involves recognition of it’s Trp37 residue with a Guanine residue of the target nucleic acid sequences. Herein, we characterized this dynamic Trp37-G recognition with sequences involved in reverse transcription and genomic RNA packaging. Using the fluorescent thienoguanosine (thG) and 2-thienyl-3-hydroxychromone (3HCnt) nucleoside analogues, we determined the whole set of kinetic rate constants for annealing of (-)PBS with (+)PBS in the absence and presence of NC. We also investigated the role of NA sugar in NC-RNA and NC-DNA complexes, as NC binds with opposite polarity to DNA and RNA sequences. We confirmed that the interaction of the Trp37 residue with guanines was critical for the formation of complexes with both RNA and DNA variants of PBS and SL3. Finally, we performed screening of NC inhibitors and tested the selected hits on a thG-based assay.
129

Taxonomy and Symbiosis in Associations of Physciaceae and Trebouxia / Taxonomie und Symbiose in Assoziationen von Physciaceen und Trebouxia

Helms, Gert 06 November 2003 (has links)
Die Familie der Physciaceen (lichenisierte Ascomyceten) und deren kompatible Photobionten wurden mit Hilfe von nrITS-Sequenzierungen untersucht. Es wurde Frisch- oder Herbarmaterial bearbeitet, das weltweit gesammelt worden war und 23 der 27 Physciaceengattngen repräsentierte. Die Sequenzdaten erlaubten eine differenzierte taxonomische Bearbeitung beider Biontengruppen. Basale Linien der Physciaceenphylogenie waren eng korreliert mit der Verteilung mehrerer phänotypischer Merkmale. Es konnte gezeigt werden, daß die Caliciaceen, eine andere Flechtenfamilie, die Schwestergruppe zu einer der vier Hauptlinien der Physciaceen bilden. Alle Proben der Physciaceen waren mit Algen aus der Gattung Trebouxia assoziiert. Ein Datensatz von über 300 Trebouxia nrITS-Sequenzen wurde zusammengestellt, der eine zuvor ungekannte Diversität innerhalb der Gattung Trebouxia repräsentiert. Die Taxonomie dieser Gattung wurde revidiert und ein System zur Abgrenzung und Zuordnung von nrITS-Varianten vorgeschlagen, das eine Strukturierung der gefundenen Diversität erlaubt. Viele der untersuchten Physciaceenarten erschienen hoch selektiv in Bezug auf ihre kompatiblen Photobionten. Im Gegensatz dazu konnte bei keinem der Photobionten eine Beschränkung auf nur eine Mycobiontenlinie gezeigt werden. Die Beschränkung vieler Mycobionten auf einen bestimmten Photobionten wurde als eine ökologische Abhängigkeit des Mycobionten von seinem kompatiblen Photobionten interpretiert. Daher wurde untersucht, ob Artbildungsereignisse in Trebouxia, Artbildungsereignisse in den assoziierten Physciaceen auslösen können. In einem Vergleich der Trebouxia- mit der Physciaceenphylogenie konnten jedoch keine korrelierten Verzweigungsmuster festgestellt werden. Hauptlinien der Trebouxien waren allerdings mit Umweltparametern, wie z.B. Substrat-pH und Makroklima korreliert. Die Evolution der Physciaceen war von diesen Faktoren offensichtlich deutlich weniger abhängig.Die nrSSU-Gene der Physciaceen enthielten mehr Introns als die aller anderen bekannter Organismengruppen. Der einzigartige Datensatz konnte genutzt werden, um konservierte Regionen innerhalb dieser Introns zu identifizieren. Auf diese konservierten Regionen konnten Primer konstruiert werden, die mit allen Introns einer Insertionsstelle kompatibel waren. Mit Hilfe dieser Primer konnten Introns detektiert werden, die bei der nrSSU-Sequenzierung unerkannt geblieben waren.
130

The use of Gibson Assembly for DNA cloning / Användning av Gibson Assembly för att klona DNA

Johansson, Samuel January 2022 (has links)
This thesis report revolved around the cloning process of plasmids. Attempts of cloning the red fluorescent protein mCherry, and the green fluorescent protein EGFP from various plasmids, into other plasmids containing different cell-junction/cytoskeleton plasmids were made. These plasmids were first amplified using PCR, and then cloned using Gibson-Assembly, and then transfected into live HEK293T or MDCK-II cells. After the transfection, the cells were examined in a microscope. The results showed no signal or localization for the cloned plasmids in their respective corresponding channel, 561 nm for the red fluorescent protein mCherry or 488 nm for the green fluorescent protein EGFP. The step that went wrong was the PCR step in the cloning process, since the backbone vector was not successfully amplified. The reasons for this was either that the backbone vector was too long, the primers regions were to rich with Guanine and Cytoseine, or the primers being too long. / Den här tesen kretsade kring kloningsprocessen för plasmider. Det gjordes försök att från plasmider klona in det röda fluorescerande proteinet mCherry, samt det gröna fluorescerande proteinet EGFP in i andra plasmider som innehöll olika cell-junction proteiner. Både det fluorescerande fragmenten och plasmid-vektorerna innehållande cell-junction proteinerna amplifierades med PCR. Sedan gjordes Gibson-Assembly som var själva kloningsmetoden. Efter det transfekterades HEK293T, samt MDCK-II celler med lösningen från Gibson-Assembly kloningen. Dessa celler undersöktes sedan i mikroskop. Resultatet visade inga tydliga signaler varken i 561 nm kanalen (mCherry), eller i 488 nm kanalen (EGFP), vilket betyder att kloningen inte fungerade. Steget som gick fel var PCR-steget i själva kloningsprocessen, då plasmid-vektorerna inte amplifierades. Anledningen till detta var antingen att själva plasmid-vektorerna var för långa, primer regionerna hade för mycket Guanin och Cytosin, eller att alla primers själva var för långa.

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