XML-tekniikoihin perustuva ajurimalli LabView-pohjaiseen testausympäristöön

XML-muotoista tiedonesitystapaa hyödynnetään yhä enemmän esitettäessä rakenteellista tietoa. Tarkoituksena on antaa yleishyödyllinen ja uudelleenkäytettävä tapa jakaa yleistä tietoa erilaisten rajapintojen yli. XML-tekniikoita käytetään myös korjaamaan aiemmin tehdyissä sovellutuksissa esiintyneitä puutteita ja parantamaan niiden toimintaa. Tässä diplomityössä esitellään Telestelle LabView-pohjaiseen testaussovellusympäristöön suunniteltava ajuriuudistus. Työssä paranneltiin aiempaa ajurimallia soveltamalla siihen XML-tekniikoita hyödyntäviä toimintoja. Tarkoituksena oli vähentää testaussovelluskehityksessä vaadittavaa ohjelmointityötä korvaamalla sovelluksiin kovakoodatut ominaisuudet XML-pohjaisilla konfiguraatiotiedostoilla. Järjestelmän pohjana on yleiskäyttöinen ajuri, joka käyttää Telesten omaa EMS-protokollaa kommunikoinnissaan testattavien tuotteiden kanssa. Ajurimalli käyttää XML-pohjaisia konfiguraatiotiedostoja määrittelemään testattavien tuotteiden ominaisuuksia. XML-skeematiedostoilla esitetään ajurin käyttämän kommunikaatioprotokollan viestityypit ja niiden rakenteet. Työn tuloksena onnistuttiin luomaan uudenlainen XML-tekniikoita hyödyntävä ajurimalli. Yhteen yhteiseen ajuriin perustuva malli yhdenmukaistaa testaussovelluksien toteuttamista ja vähentää tarvittavaa ohjelmointityötä. Ajurin käyttöä helpotettiin toteuttamalla testaussovelluksien kehitysympäristöön erityinen editori, jolla voidaan helposti luoda ajuria käyttäviä toimintoja.

Evolution of Bioaffinity Reagents by Phage Display

Antibodies are natural binding proteins produced in vertebrates as a response to invading pathogens and foreign substances. Because of their capability for tight and specific binding, antibodies have found use as binding reagents in research and diagnostics. Properties of cloned recombinant antibodies can be further improved by means of in vitro evolution, combining mutagenesis with subsequent phage display selection. It is also possible to isolate entirely new antibodies from vast naïve or synthetic antibody libraries by phage display. In this study, library techniques and phage display selection were applied in order to optimise binding scaffolds and antigen recognition of antibodies, and to evolve new and improved bioaffinity reagents. Antibody libraries were generated by random and targeted mutagenesis. Expression and stability were mainly optimised by the random methods whereas targeted randomisation of the binding site residues was used for optimising the binding properties. Trinucleotide mutagenesis allowed design of defined randomisation patterns for a synthetic antibody library. Improved clones were selected by phage display. Capture by a specific anti- DHPS antibody was exploited in the selection of improved phage display of DHPS. Efficient selection for stability was established by combining phage display selection with denaturation under reducing conditions. Broad-specific binding of a generic anti-sulfonamide antibody was improved by selection with one of the weakest binding sulfonamides. In addition, p9 based phage display was studied in affinity selection from the synthetic library. A TIM barrel protein DHPS was engineered for efficient phage display by combining cysteinereplacement with random mutagenesis. The resulting clone allows use of phage display in further engineering of DHPS and possibly use as an alternative-binding scaffold. An anti-TSH scFv fragment, cloned from a monoclonal antibody, was engineered for improved stability to better suite an immunoassay. The improved scFv tolerates 8 – 9 °C higher temperature than the parental scFv and should have sufficient stability to be used in an immunoanalyser with incubation at 36 °C. The anti-TSH scFv fragment was compared with the corresponding Fab fragment and the parental monoclonal antibody as a capturing reagent in a rapid 5-min immunoassay for TSH. The scFv fragment provided some benefits over the conventionally used Mab in anayte-binding capacity and assay kinetics. However, the recombinant Fab fragment, which had similar kinetics to the scFv, provided a more sensitive and reliable assay than the scFv. Another cloned scFv fragment was engineered in order to improve broad-specific recognition of sulfonamides. The improved antibody detects different sulfonamides at concentrations below the maximum residue limit (100 μg/kg in EU and USA) and allows simultaneous screening of different sulfonamide drug residues. Finally, a synthetic antibody library was constructed and new antibodies were generated and affinity matured entirely in vitro. These results illuminate the possibilities of phage display and antibody engineering for generation and optimisation of binding reagents in vitro and indicate the potential of recombinant antibodies as affinity reagents in immunoassays.

The structures of the honeydew oligosaccharides synthesized by Claviceps africana

The structures of the principal oligosaccharides in the honeydew exudate of the sorghum ergot pathogen Claviceps africana, which has become epidemic in the Americas, have been characterized through linkage analysis using FAB-MS and GC-MS techniques, as 1,6-di-b-D-fructofuranosyl-D-mannitol and 1,5-di-b-D-fructofuranosyl-D-arabitol trisaccharides, 1-b-D-fructofuranosyl-D-mannitol and 5-b-D-fructofuranosyl-D-arabitol disaccharides and other minor disaccharides and trisaccharides. Their structural diversity is explained according to perceived biosynthetic interrelationships in pathways that appear to be unique amongst ergot fungi, particularly concerning intra-molecular reduction of fructose. The oligosaccharide, 1,6-di-b-D-fructofuranosyl-D-mannitol, which inhibits C. africana macrospore germination at a concentration in water of 1 g/mL or more, forms together with other slightly less bioactive oligosaccharides, the basis of a novel strategy to limit ergot disease losses in hybrid sorghum seed production.

Caracterização de dois equipamentos de beneficiamento e classificação de tomates para mesa

No Brasil, nos últimos anos, tem aumentado o beneficiamento e a classificação de frutas e hortaliças em equipamentos. O objetivo deste trabalho foi caracterizar a etapa de limpeza de dois equipamentos de beneficiamento de tomate de mesa, um nacional e outro importado, instalados na região metropolitana de Campinas - SP. A caracterização foi realizada quanto ao número, tipo e velocidade de rotação das escovas, aplicação de água, tempo de permanência dos frutos e eficiência de limpeza. O tempo de permanência foi mensurado com relação ao tamanho dos frutos, segundo os padrões da CEAGESP. Para mensurar a eficiência, foi desenvolvido um equipamento composto de um anel deslizante envolvido por um pano branco. As amostragens foram realizadas antes e após a etapa de limpeza e avaliadas utilizando colorímetro HUNTER Lab. Por meio dos resultados, observaram-se diferenças entre os dois equipamentos, em que o importado apresentou menor número de escovas, rotações mais baixas e maior consumo de água que o nacional. No equipamento nacional, o tempo de permanência dependeu do tamanho dos frutos, não sendo encontrada essa correlação no equipamento importado, e ambos tiveram a mesma eficiência de limpeza. Conclui-se que a limpeza está relacionada à interação entre os parâmetros estudados, sendo também necessário realizar manutenções nos equipamentos das unidades para a melhoria no funcionamento.

Portable flow board for storage of fruits and vegetables in mini-chambers with controlled atmosphere

ABSTRACT A portable flow board system was developed in the present study with the aim to facilitate lab-scale experiments of controlled atmosphere (CA) with fruits and vegetables. This sturdy flow board combines ease fabrication, low cost and gas economy. Its functionality is provided by manifolds and gas mixers. Each gaseous component is supplied by a gas cylinder through a differential valve of adjusted pressure control, generally at 6 kPa, and forced through 13 standardized restrictors coupled to each manifold output. Controlled atmospheres are then formed with one, two or three gases in 13 gas mixers affixed to the flow board base, which are further conducted through flexible tubes to storage mini-chambers that can also be used to study metabolic consumption and production of gaseous components. The restrictors used in the flow gaseous components were manufactured from microhematocrit test-type capillary glass tubes following the hot forming method under continuous air flow. The portable flow board showed to be low cost and simple post-harvest equipment that allows preparing controlled atmospheres in open systems with stable composition and flow, in a manner similar to traditional flow boards with control of gas escape by barostats.

Incidência de fístulas digestivas nos ferimentos toracoabdominais

Os ferimentos penetrantes localizados na transição toracoabdominal (FTA), além da dificuldade diagnóstica, merecem especial atenção em relação à conduta adotada para o controle das complicações abdominais. Os autores analisaram 110 pacientes atendidos na Disciplina de Cirurgia do Trauma do Departamento de Cirurgia da UNICAMP, de 1988 a 1998, apresentando ferida penetrante toracoabdominal e submetidos à laparotomia exploradora com drenagem pleural fechada. As fístulas digestivas foram estudadas quanto à incidência, ao tratamento e à evolução pós-operatória. Do total de pacientes, 91 (82,7%) eram do sexo masculino e 19 (17,3%) do sexo feminino. A faixa etária situou-se entre 13 e 63 anos. Os FTA foram causados por projétil de arma de fogo (PAF) em 60 (54,5%) casos e por arma branca (FAB) em 50 (45,5%). As fístulas digestivas ocorreram em seis (5,4%) dos pacientes estudados, sendo quatro (3,6%) casos de fístula pancreática, um (0,9%) de fístula gástrica e um (0,9%) de fístula biliar, todos tratados de maneira conservadora, apresentando evolução favorável com resolução espontânea.

O impacto econômico do trauma em um hospital universitário

OBJETIVOS: Determinar o impacto financeiro do trauma em um hospital universitário em Curitiba. MÉTODO: Estudo retrospectivo de pacientes vítimas de trauma, internados no Hospital Universitário Cajuru, divididos de acordo com o mecanismo de trauma. Foram analisados o número de dias de internamento e o custo assistencial total de cada paciente declarados na ficha de Autorização de Internação Hospitalar (AIH). A partir daí, calculou-se o custo médio, a média de dias de internamento e o custo médio paciente/dia. RESULTADOS: O custo médio de 349 pacientes estudados foi de US$ 568,22; das vítimas por ferimento por arma de fogo (FAF), US$ 692,50; das vítimas de ferimento de arma branca (FAB), US$ 676,9; das vítimas de agressão, US$ 412,3; das vítimas de queda, US$ 503,05 e das vítimas de acidente de trânsito, US$ 600,30. A média de dias de internamento da amostra total foi de 6,2; das vítimas de FAF, 7,7; das vítimas de FAB, 5,3; das vítimas de agressão, 6,7; das vítimas de queda, 7 e das vítimas de acidente de trânsito 6. O custo médio paciente/dia da amostra total foi de US$ 91,65; das vítimas de FAF, US$ 89,99; das vítimas de FAB, US$ 127,66; das vítimas de agressão, US$ 61,54; das vítimas de queda, US$ 71,86 e das vítimas de acidente de trânsito, US$ 100,05. CONCLUSÃO: Este estudo, como tantos outros, mostra que o trauma é uma doença negligenciada, importante para a saúde publica por seu grande impacto social e econômico e passível de redução através de medidas de prevenção.

Ferimentos cervicais: análise retrospectiva de 191 casos

OBJETIVOS: Analisar a epidemiologia e a conduta nos ferimentos cervicais. MÉTODO: Foram analisados 487.128 prontuários de pacientes que ingressaram no Serviço de Emergência do Hospital Universitário Cajuru no período de 01/1996 a 06/2001. Destes, selecionaram-se 378 pacientes com ferimentos cervicais. Foram excluídos 153 que apresentavam lesões associadas e 14 por óbito no atendimento inicial. O estudo foi feito , assim, em 191 pacientes com lesões cervicais exclusivas. Avaliou-se a localização da ferida, o mecanismo de trauma, o comprometimento do platisma, sinais e sintomas, a hora de admissão e a conduta empregada. RESULTADOS: Cento e sessenta e quatro (86%) pacientes eram masculinos. A média de idade foi de 28 anos (10-72). Noventa (47%) ferimentos foram por arma de fogo (FAF) e 88 (46%) por arma branca (FAB). O principal horário de admissão foi entre 20 e 04 horas. Quanto à localização, 53% das lesões foram à esquerda, 45% à direita e 2% medianos; 36% em zona I, 55% em zona II e 9% em zona III. Em 101 o ferimento penetrou o platisma: cinqüenta e um (50%) apresentaram sinais e sintomas clínicos e receberam conduta operatória. As lesões vasculares foram as mais encontradas (20). Houve 24 (47%) cervicotomias não-terapêuticas. O tratamento conservador foi empregado em 41 (45%) casos de acordo com os exames físico e complementares. CONCLUSÕES: Homens jovens são mais acometidos quanto aos ferimentos cervicais. Estes ocorrem mais freqüentemente na zona II, e a incidência dos FAF e FAB foi equivalente. É adequado um manejo mais seletivo em relação aos ferimentos cervicais, devendo o manejo da zona II adequar-se à disposição de recursos dos serviços de trauma.

Janela pericárdica transdiafragmática no diagnóstico de lesão cardíaca

OBJETIVO: Avaliar a eficácia e as complicações da utilização da janela pericárdica (JP) transdiafragmática no diagnóstico de lesão cardíaca por ferimento penetrante. MÉTODO: No período de janeiro de 1994 a dezembro de 2004 a JP foi indicada em 245 casos com suspeita de trauma cardíaco por ferimento penetrante. Em 38 deles (15,5%) a JP foi realizada através de acesso transdiafragmático, constituindo a população do presente estudo. RESULTADOS: O mecanismo de lesão foi ferimento por projétil de arma de fogo (FPAF) em 26 casos (68,4%) e por arma branca (FAB) em 12 casos (31,6%). Na maioria dos pacientes (27 casos - 71%) os ferimentos eram múltiplos. A média de RTS foi de 7,32 e a média de PATI foi de 9,8. A JP foi considerada positiva em seis casos (15,8%) dos quais cinco (83%) por FPAF e um (17%) por FAB. Houve um paciente, vítima de múltiplos FPAF, com JP positiva e toracotomia em que não foi identificada lesão cardíaca. A sensibilidade do método foi de 97,4% e a especificidade de 100%. Em 12 (31,6%) pacientes houve associação de lesão com perfuração de víscera oca. Houve um único caso (2,6%) de complicação diretamente relacionada ao procedimento, em uma vítima de múltiplos FPAF com lesão perfurada de fígado, estômago e cólon, que evoluiu com pericardite, necessitando posteriormente de drenagem pericárdica, com boa resolução. A mortalidade foi de 7,9% (três casos), um dos quais em pós-operatório de reparo de lesão cardíaca por coagulopatia/acidose. CONCLUSÕES: A JP trandiafragmática é um procedimento que permite o rápido diagnóstico de lesão cardíaca em pacientes nos quais a prioridade é a laparotomia. Trata-se de método de fácil realização e alta sensibilidade.

Paper for printed electronics and functionality

Mass-produced paper electronics (large area organic printed electronics on paper-based substrates, “throw-away electronics”) has the potential to introduce the use of flexible electronic applications in everyday life. While paper manufacturing and printing have a long history, they were not developed with electronic applications in mind. Modifications to paper substrates and printing processes are required in order to obtain working electronic devices. This should be done while maintaining the high throughput of conventional printing techniques and the low cost and recyclability of paper. An understanding of the interactions between the functional materials, the printing process and the substrate are required for successful manufacturing of advanced devices on paper. Based on the understanding, a recyclable, multilayer-coated paper-based substrate that combines adequate barrier and printability properties for printed electronics and sensor applications was developed in this work. In this multilayer structure, a thin top-coating consisting of mineral pigments is coated on top of a dispersion-coated barrier layer. The top-coating provides well-controlled sorption properties through controlled thickness and porosity, thus enabling optimizing the printability of functional materials. The penetration of ink solvents and functional materials stops at the barrier layer, which not only improves the performance of the functional material but also eliminates potential fiber swelling and de-bonding that can occur when the solvents are allowed to penetrate into the base paper. The multi-layer coated paper under consideration in the current work consists of a pre-coating and a smoothing layer on which the barrier layer is deposited. Coated fine paper may also be used directly as basepaper, ensuring a smooth base for the barrier layer. The top layer is thin and smooth consisting of mineral pigments such as kaolin, precipitated calcium carbonate, silica or blends of these. All the materials in the coating structure have been chosen in order to maintain the recyclability and sustainability of the substrate. The substrate can be coated in steps, sequentially layer by layer, which requires detailed understanding and tuning of the wetting properties and topography of the barrier layer versus the surface tension of the top-coating. A cost competitive method for industrial scale production is the curtain coating technique allowing extremely thin top-coatings to be applied simultaneously with a closed and sealed barrier layer. The understanding of the interactions between functional materials formulated and applied on paper as inks, makes it possible to create a paper-based substrate that can be used to manufacture printed electronics-based devices and sensors on paper. The multitude of functional materials and their complex interactions make it challenging to draw general conclusions in this topic area. Inevitably, the results become partially specific to the device chosen and the materials needed in its manufacturing. Based on the results, it is clear that for inks based on dissolved or small size functional materials, a barrier layer is beneficial and ensures the functionality of the printed material in a device. The required active barrier life time depends on the solvents or analytes used and their volatility. High aspect ratio mineral pigments, which create tortuous pathways and physical barriers within the barrier layer limit the penetration of solvents used in functional inks. The surface pore volume and pore size can be optimized for a given printing process and ink through a choice of pigment type and coating layer thickness. However, when manufacturing multilayer functional devices, such as transistors, which consist of several printed layers, compromises have to be made. E.g., while a thick and porous top-coating is preferable for printing of source and drain electrodes with a silver particle ink, a thinner and less absorbing surface is required to form a functional semiconducting layer. With the multilayer coating structure concept developed in this work, it was possible to make the paper substrate suitable for printed functionality. The possibility of printing functional devices, such as transistors, sensors and pixels in a roll-to-roll process on paper is demonstrated which may enable introducing paper for use in disposable “onetime use” or “throwaway” electronics and sensors, such as lab-on-strip devices for various analyses, consumer packages equipped with product quality sensors or remote tracking devices.

Fifty years in the blink of an eye: a retrospective study of ocular and periocular lesions in domestic animals

A survey was undertaken aiming to obtain an overview of ocular and periocular lesions diagnosed in domestic mammals over a period of 50 years in a veterinary pathology diagnostic laboratory in the Central Region of the State of Rio Grande do Sul, Brazil. In this lab, 33,075 histophatological exams had been performed over the period surveyed, of which 540 (1.6%) concerned ocular and periocular lesions. For various reasons ninety specimens were excluded from the study and the remaining 450 consisted of samples from dogs (53.5%), cattle (28.2%), cats (11.1%), horses (5.1%) sheep (1.3%), rabbits (0.4%), and pig (0.2%). The eyelids were the most prevalent (248/450) site of lesions in each of the species studied, followed by third eyelid (73/450), and conjunctiva (27/450). In dogs (241 samples) lesions in sebaceous glands (including Meibomian glands) were the most common findings (75/241), followed by melanocytic tumors (52/241) and nonspecific conjunctivitis (13/241). Squamous cell neoplasms, both benign and malignant, were relatively common. In cattle, anatomical sites affected by ocular and periocular lesions, in decreasing order of frequency, were eyelid, cornea and third eyelid. Squamous cell carcinoma (SCC) alone accounted for 80.3% of all diagnoses, while all neoplastic lesions made up for 85.0% of the lesions diagnosed in cattle. Neoplasia accounted for most of the lesions diagnosed in cats (39/50 cases); all of these were malignant, and SCC, hemangiosarcoma and fibrosarcoma were the most common types diagnosed. In horses, 19 out of 23 submissions were neoplasms and most were sarcoid (8/23) and SCC (8/23). There were six submissions from sheep with unpigmented skin, all of which represented SCC of the eyelids (5) and third eyelid (1).

Characterisation of waste for combustion : with special reference to the role of zinc

Waste combustion has gone from being a volume reducing discarding-method to an energy recovery process for unwanted material that cannot be reused or recycled. Different fractions of waste are used as fuel today, such as; municipal solid waste, refuse derived fuel, and solid recovered fuel. Furthermore, industrial waste, normally a mixture between commercial waste and building and demolition waste, is common, either as separate fuels or mixed with, for example, municipal solid waste. Compared to fossil or biomass fuels, waste mixtures are extremely heterogeneous, making it a complicated fuel. Differences in calorific values, ash content, moisture content, and changing levels of elements, such as Cl and alkali metals, are common in waste fuel. Moreover, waste contains much higher levels of troublesome trace elements, such as Zn, which is thought to accelerate a corrosion process. Varying fuel quality can be strenuous on the boiler system and may cause fouling and corrosion of heat exchanger surfaces. This thesis examines waste fuels and waste combustion from different angles, with the objective of giving a better understanding of waste as an important fuel in today’s fuel economy. Several chemical characterisation campaigns of waste fuels over longer time periods (10-12 months) was used to determine the fossil content of Swedish waste fuels, to investigate possible seasonal variations, and to study the presence of Zn in waste. Data from the characterisation campaigns were used for thermodynamic equilibrium calculations to follow trends and determine the effect of changing concentrations of various elements. The thesis also includes a study of the thermal behaviour of Zn and a full—scale study of how the bed temperature affects the volatilisation of alkali metals and Zn from the fuel. As mixed waste fuel contains considerable amounts of fresh biomass, such as wood, food waste, paper etc. it would be wrong to classify it as a fossil fuel. When Sweden introduced waste combustion as a part of the European Union emission trading system in the beginning of 2013 there was a need for combustion plants to find a usable and reliable method to determine the fossil content. Four different methods were studied in full-scale of seven combustion plants; 14Canalysis of solid waste, 14C-analysis of flue gas, sorting analysis followed by calculations, and a patented balance method that is using a software program to calculate the fossil content based on parameters from the plant. The study showed that approximately one third of the coal in Swedish waste mixtures has fossil origins and presented the plants with information about the four different methods and their advantages and disadvantages. Characterisation campaigns also showed that industrial waste contain higher levels of trace elements, such as Zn. The content of Zn in Swedish waste fuels was determined to be approximately 800 mg kg-1 on average, based on 42 samples of solid waste from seven different plants with varying mixtures between municipal solid waste and industrial waste. A review study of the occurrence of Zn in fuels confirmed that the highest amounts of Zn are present in waste fuels rather than in fossil or biomass fuels. In tires, Zn is used as a vulcanizing agent and can reach concentration values of 9600-16800 mg kg-1. Waste Electrical and Electronic Equipment is the second Zn-richest fuel and even though on average Zn content is around 4000 mg kg-1, the values of over 19000 mg kg-1 were also reported. The increased amounts of Zn, 3000-4000 mg kg-1, are also found in municipal solid waste, sludge with over 2000 mg kg-1 on average (some exceptions up to 49000 mg kg-1), and other waste derived fuels (over 1000 mg kg-1). Zn is also found in fossil fuels. In coal, the average level of Zn is 100 mg kg-1, the higher amount of Zn was only reported for oil shale with values between 20-2680 mg kg-1. The content of Zn in biomass is basically determined by its natural occurrence and it is typically 10-100 mg kg-1. The thermal behaviour of Zn is of importance to understand the possible reactions taking place in the boiler. By using thermal analysis three common Zn-compounds were studied (ZnCl2, ZnSO4, and ZnO) and compared to phase diagrams produced with thermodynamic equilibrium calculations. The results of the study suggest that ZnCl2(s/l) cannot exist readily in the boiler due to its volatility at high temperatures and its conversion to ZnO in oxidising conditions. Also, ZnSO4 decomposes around 680°C, while ZnO is relatively stable in the temperature range prevailing in the boiler. Furthermore, by exposing ZnO to HCl in a hot environment (240-330°C) it was shown that chlorination of ZnO with HCl gas is possible. Waste fuel containing high levels of elements known to be corrosive, for example, Na and K in combination with Cl, and also significant amounts of trace elements, such as Zn, are demanding on the whole boiler system. A full-scale study of how the volatilisation of Na, K, and Zn is affected by the bed temperature in a fluidised bed boiler was performed parallel with a lab-scale study with the same conditions. The study showed that the fouling rate on deposit probes were decreased by 20 % when the bed temperature was decreased from 870°C to below 720°C. In addition, the lab-scale experiments clearly indicated that the amount of alkali metals and Zn volatilised depends on the reactor temperature.

Methods of genetic diversity creation and functional display for directed evolution experiments

Protein engineering aims to improve the properties of enzymes and affinity reagents by genetic changes. Typical engineered properties are affinity, specificity, stability, expression, and solubility. Because proteins are complex biomolecules, the effects of specific genetic changes are seldom predictable. Consequently, a popular strategy in protein engineering is to create a library of genetic variants of the target molecule, and render the population in a selection process to sort the variants by the desired property. This technique, called directed evolution, is a central tool for trimming protein-based products used in a wide range of applications from laundry detergents to anti-cancer drugs. New methods are continuously needed to generate larger gene repertoires and compatible selection platforms to shorten the development timeline for new biochemicals. In the first study of this thesis, primer extension mutagenesis was revisited to establish higher quality gene variant libraries in Escherichia coli cells. In the second study, recombination was explored as a method to expand the number of screenable enzyme variants. A selection platform was developed to improve antigen binding fragment (Fab) display on filamentous phages in the third article and, in the fourth study, novel design concepts were tested by two differentially randomized recombinant antibody libraries. Finally, in the last study, the performance of the same antibody repertoire was compared in phage display selections as a genetic fusion to different phage capsid proteins and in different antibody formats, Fab vs. single chain variable fragment (ScFv), in order to find out the most suitable display platform for the library at hand. As a result of the studies, a novel gene library construction method, termed selective rolling circle amplification (sRCA), was developed. The method increases mutagenesis frequency close to 100% in the final library and the number of transformants over 100-fold compared to traditional primer extension mutagenesis. In the second study, Cre/loxP recombination was found to be an appropriate tool to resolve the DNA concatemer resulting from error-prone RCA (epRCA) mutagenesis into monomeric circular DNA units for higher efficiency transformation into E. coli. Library selections against antigens of various size in the fourth study demonstrated that diversity placed closer to the antigen binding site of antibodies supports generation of antibodies against haptens and peptides, whereas diversity at more peripheral locations is better suited for targeting proteins. The conclusion from a comparison of the display formats was that truncated capsid protein three (p3Δ) of filamentous phage was superior to the full-length p3 and protein nine (p9) in obtaining a high number of uniquely specific clones. Especially for digoxigenin, a difficult hapten target, the antibody repertoire as ScFv-p3Δ provided the clones with the highest affinity for binding. This thesis on the construction, design, and selection of gene variant libraries contributes to the practical know-how in directed evolution and contains useful information for scientists in the field to support their undertakings.

Potencial de espécies vegetais para a remediação do herbicida trifloxysulfuron-sodium

Este trabalho teve como objetivo avaliar a eficiência de espécies vegetais na remediação do herbicida trifloxysulfuron-sodium em solos, utilizando o feijão (Phaseolus vulgaris) como planta indicadora. Os tratamentos foram compostos pela combinação entre as espécies Calopogonium mucunoides, Crotalaria juncea, Crotalaria spectabilis, Vicia sativa, Cajanus cajan, Canavalia ensiformis, Medicago sativa, Dolichus lab lab, Penisetum glaucum, Stylosantes guianensis, Mucuna deeringiana, Mucuna cinereum, Mucuna aterrima, Raphanus sativus e Lupinus albus. Todas as espécies foram semeadas em vasos no dia seguinte à aplicação do trifloxysulfuron-sodium em três doses (0,00; 3,75; e 15,00 g ha-1). Após 80 dias da semeadura, as espécies vegetais foram cortadas na altura do coleto e a parte aérea destas descartada. A seguir, foi realizada a semeadura do feijão (cultivar Pérola). Aos 45 dias após a emergência das plantas de feijão avaliaram-se a altura e a massa seca da parte aérea das plantas. Melhor eficiência na descontaminação do trifloxysulfuron-sodium em solo foi obtida pelas espécies M. aterrima e C. ensiformis.