Evolución dirigida de proteínas: La evolución dirigida es una técnica que nos permite explorar funciones enzimáticas que no son requeridas en el ambiente natural. Esta técnica, simula procesos genéticos naturales y de selección. Esta estrategia se utiliza cuando un diseño racional es muy complicado. Consiste en una repetición de ciclos de diversificación y selección que llevan a la acumulación de mutaciones benéficas. Aquí se presenta dos ejemplos de evolución dirigida con los cuales se ha trabajado directamente: la ADN polimerasa del organismo  Thermus aquaticus usada comúnmente en PCR, y la proteína LacI que regula la expresión de genes usados para el metabolismo de lactosa en E. Coli. / Directed evolution allows us to explore protein functionalities not required in the natural environment. It mimics natural genetic processes and selective pressures. This approach is used when the molecular basis is not completely understood and rational design is a difficult task. This approach consists of serial cycles of consecutive diversification and selection which eventually lead to the accumulation of beneficial mutations. Here are presented two cases where directed evolution is used to modify two different proteins: Taq polymerase, enzyme used for DNA extension in PCR, and the LacI repressor protein which regulates gene expression on E.coli.

Submitted by FLAVIA ARANTES TAPARO null (flavia-arantes@hotmail.com) on 2016-04-08T19:33:11Z No. of bitstreams: 1 Dissertação_Mestrado em Educação_Flávia Arantes Táparo.pdf: 966051 bytes, checksum: 3e26dca0c17b0f77a7021a0e53ff2728 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-04-11T17:49:01Z (GMT) No. of bitstreams: 1 taparo_fa_me_mar.pdf: 966051 bytes, checksum: 3e26dca0c17b0f77a7021a0e53ff2728 (MD5) / Made available in DSpace on 2016-04-11T17:49:01Z (GMT). No. of bitstreams: 1 taparo_fa_me_mar.pdf: 966051 bytes, checksum: 3e26dca0c17b0f77a7021a0e53ff2728 (MD5) Previous issue date: 2016-03-09 / Alterações comportamentais e ocorrência de acidentes podem causar diversos problemas para o desenvolvimento infantil, mas são escassos trabalhos que investiguem estas variáveis de modo integrado, na busca de indicadores para futura atuação. Este estudo teve como objetivos caracterizar o comportamento e a ocorrência de acidentes de escolares do 5º ano do ensino fundamental e verificar as relações entre ambas as variáveis e entre os dados do comportamento obtidos com diferentes informantes. A pesquisa foi realizada em uma escola da rede municipal de Ensino Fundamental de uma cidade do interior do estado de São Paulo. Participaram 60 alunos de quatro turmas de 5º ano, de ambos os sexos e média de idade de dez anos, seus respectivos responsáveis e as quatro professoras das turmas. Foram utilizados o Questionário de Capacidades e Dificuldades (SDQ) com responsáveis e professoras e o Questionário de Rastreamento de Acidentes (QRA) com os escolares, mediante Termo de Consentimento. Verificou-se predominância de perfil normal de escolares de ambos os sexos em todas as escalas do SDQ, tanto de dificuldades quanto de capacidades. Entretanto, houve padrão anormal de comportamentos, segundo os responsáveis, na escala de sintomas emocionais, com 26,9% para o sexo feminino e 38,1% para o masculino e, de acordo com as professoras, nas escalas de problemas de conduta e de hiperatividade, com 37,5% cada. Foi identificada a ocorrência de 1315 acidentes, predominando acidentes de bicicleta, causados por contato com material cortante e por impacto contra objetos, diferente da literatura, que aponta predominância das quedas. Foi observado maior número de acidentes para o sexo feminino, diferente da literatura. Não foi observada diferença estatisticamente significante na comparação dos perfis comportamentais dos escolares. Entretanto, foi observada diferença significativa no acidente causado por contato com material cortante. Nas respostas dos responsáveis e das professoras ao SDQ, houve correlação estatisticamente significante em quatro situações: 1) problemas de conduta segundo as professoras e os responsáveis; 2) problemas de conduta segundo as professoras e hiperatividade segundo os responsáveis; 3) hiperatividade segundo as professoras e segundo os responsáveis; e 4) comportamento pró social segundo as professoras e hiperatividade segundo os responsáveis. Nas análises de correlação entre as respostas dos responsáveis ao SDQ e das crianças ao QRA e entre as respostas das professoras ao SDQ e das crianças ao QRA predominantemente não houve correlação estatisticamente significativa, diferente da literatura. Foi observada correlação negativa entre o comportamento de hiperatividade das crianças, segundo as professoras, e o acidente de bicicleta e entre o comportamento de sintomas emocionais nas crianças, segundo as professoras, e o acidente do tipo impacto contra objetos. Concluiu-se que o perfil comportamental dos escolares esteve predominantemente dentro dos padrões de normalidade, que a ocorrência de acidentes foi importante e houve pouca correlação significativa entre os resultados, diferentemente da literatura. Sugeriu-se ampliação da amostra e de locais de coletas para investigar os resultados peculiares. / Children's behavioral changes or accidents in childhood can be particularly troublesome for child development, but there are scarce studies that investigate these variables in an integrated manner, seeking indicators for future practice. This study aimed to characterize the behavior and the occurrence of accidents with 5th year of elementary school students and examine relationships between both variables and between the behavior's data from different informants. The survey was conducted in a municipal elementary school of a medium-sized city in the state of São Paulo. Attended by 60 students from four classes of 5th grade, of both sexes and an averege age of ten years old, their family and the four teachers of the classes. The Strengths and Difficulties Questionnaire (SDQ) was applied with family members and teachers and Accident Tracking Survey (QRA) was applied with the students; the participation of all was consented by signing the Term of Consent. There was a predominance of normal profile of students of both sexes in all SDQ scales, both in difficulties and in capabilities. However, there was an unusual standard of behavior, according to those responsible, in the emotional symptoms scale, with 26.9% for females and 38.1% for male and, according to the teachers, the scales of conduct problems and hyperactivity, with 37.5% each. The occurrence of 1315 accidents was identified, being bicycle accidents, contact with sharp materials and impact against objects, different from the literature, which indicates predominance in falls. There was a higher number of accidents for females, unlike literature. There was no statistically significant difference when comparing the behavioral profiles of schools. However, a significant difference was observed in the accident caused by contact with sharps materials. About the answers from their legal guardians and teachers of the SDQ, there was a statistically significant correlation in four situations: 1) conduct problems according to teachers and those responsible; 2) conduct problems according to teachers and hyperactivity, according to responsible; 3) hyperactivity according to the teachers and the responsible; and 4) social pro behavior according to the teachers and hyperactivity according to those responsible. In the correlation analysis between the answers of those responsible to the SDQ and children to the QRA and between the answers of teachers to SDQ and children to QRA predominantly there was no statistically significant correlation, unlike literature. Negative correlation between children's hyperactive behavior was observed, according to the teachers, and the bicycle accident and between the behavior of emotional symptoms in children, according to the teachers, and the accident of the type of impact against objects. We conclude that the behavioral profile of the students was mostly within the normal range, the occurrence of accidents was important and there was little correlation between the results, unlike literature. It is suggested larger sample collections and locations to investigate the peculiar results.

Submitted by Rafaelle Batistella Pires (rafaelle.pires@gmail.com) on 2018-03-07T15:41:30Z No. of bitstreams: 1 Tese Rafaelle Batistella Pires.pdf: 2978722 bytes, checksum: f79b8076fd69934911d1a338cd131aa3 (MD5) / Approved for entry into archive by Luciana Pizzani null (luciana@btu.unesp.br) on 2018-03-08T20:07:06Z (GMT) No. of bitstreams: 1 pires_rb_dr_bot.pdf: 2978722 bytes, checksum: f79b8076fd69934911d1a338cd131aa3 (MD5) / Made available in DSpace on 2018-03-08T20:07:06Z (GMT). No. of bitstreams: 1 pires_rb_dr_bot.pdf: 2978722 bytes, checksum: f79b8076fd69934911d1a338cd131aa3 (MD5) Previous issue date: 2018-02-20 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A Síndrome do Desconforto Respiratório Agudo (SDRA) cursa com alta morbi-mortalidade apesar dos avanços no entendimento de sua fisiopatologia e tratamento. A terapia ventilatória baseia-se na proteção pulmonar, sendo a ventilação oscilatória de alta frequência (VOAF) uma opção de método protetor. A posição prona (PP) é terapia adjuvante que possibilita homogeneização da distribuição do volume corrente (VC) e promove recrutamento alveolar. O objetivo do estudo foi investigar o efeito da posição prona associada à VOAF e ventilação mecânica convencional (VMC) protetora sobre a oxigenação, inflamação, dano oxidativo e histologia pulmonares, comparando-a à posição supina em ambos os modos ventilatórios. Foram instrumentados 75 coelhos com traqueostomia e acessos vasculares. A lesão pulmonar aguda (LPA) foi induzida por lavagem traqueal de salina aquecida (30mL/Kg, 38°C). Os animais foram então aleatorizados em cinco grupos (n=15): 1) GC (Controle): animais sadios em VMC protetora basal; 2) GVMS: animais com LPA em VMC protetora e posição supina; 3) GVMP: animais com LPA em VMC protetora e posição prona; 4) GVAFS: animais com LPA em VOAF e posição supina; 5) GVAFP: animais com LPA em VOAF e posição prona. Após, foram submetidos a quatro horas de VMC protetora (modo pressão regulada-volume controlado, PEEP 10 cmH2O, VC 6mL/kg, Ti 0,5s, FR 40 rpm e FiO2 1) ou VOAF (MAP 15 mmHg, FR 10Hz, amplitude 22 e FiO2 1). O nível de significância foi de 5%. Após a indução, os grupos apresentaram comportamentos semelhantes, com diminuição da relação PaO2/FiO2 e da complacência pulmonar, e aumento do índice de oxigenação (IO) e da pressão média de via aérea (p > 0,05). Ao final do experimento, houve aumento da PaO2/FiO2 nos grupos VOAF comparado aos grupos em VMC (p < 0,05). Houve queda do IO para os grupos em VOAF comparados ao GVMS (p < 0,05), porém o GVMP não diferiu deles (p > 0,05). Não houve diferença estatística quanto à contagem de células polimorfonucleares no lavado broncoalveolar (BAL) nos grupos com LPA. Não houve diferença estatística entre os grupos com lesão para a medida de TNF-alfa no plasma e para sua expressão gênica em tecido pulmonar. Entretanto, a medida de TNF-alfa no lavado broncoalveolar (BAL) e no tecido pulmonar no grupo GVMP foi menor, assemelhando-se ao controle (p > 0,05). Não houve diferença no dano oxidativo avaliado no tecido pulmonar entre os grupos (p > 0,05) e, também, na comparação entre regiões ventral e dorsal dos pulmões. O escore de lesão histológica foi menor nos grupos em VOAF, efeito potencializado no grupo em prona quando comparado aos grupos em VMC (GC = GVAFP < GVMS = GVMP), sem diferença na regionalização pulmonar. Concluimos que, em modelo de LPA por lavagem alveolar com salina aquecida em coelhos: a VOAF melhora a oxigenação quando comparados à VMC; na VMC, a PP atenua a lesão inflamatória avaliada pela medida de TNF-alfa no BAL e tecido pulmonar; os modos ventilatórios e as posições não modificam o grau de estresse oxidativo quando avaliados pelo método de malondialdeído; a VOAF melhora o escore histopatológico de lesão pulmonar, independemente da posição, mas a associação de VOAF e PP atenua a lesão histopatológica quando comparada com a VMC protetora, seja em posição prona ou supina. / Acute Respiratory Distress Syndrome (ARDS) presents with high morbidity and mortality despite advances in the understanding of its pathophysiology and treatment. Ventilatory therapy is based on the intention of injuring less, with high frequency oscillatory ventilation (HFOV) being a protective method option. Prone position (PP) is an adjuvant therapy that enables homogenization of volume tidal (VT) distribution and promotes alveolar recruitment. The aim of this study was to investigate the effects of prone position associated with HFOV and protective conventional mechanical ventilation (CMV) on oxygenation and lung inflammation, oxidative damage and histology, comparing it with the supine position in both ventilatory modes. Seventy five rabbits were submitted to tracheostomy and vascular accesses. ALI was induced by tracheal infusion of heated saline (30mL/kg, 38° C). The subjects were then ramdomized in five groups (n=15): 1) CG (Control): healthy animals in basal protective CMV; 2) MVSG: animals with ALI in protective CMV and supine position; 3) MVPG animals with ALI in protective CMV and prone position; 4) HFSG: animals with ALI in HFOV and supine position; 5) HFPG: animals with ALI in HFOV and prone position. After that, they were submitted to four hours of protective VMC (PRV mode, PEEP 10 cmH2O, VC 6ml/kg, Ti 0,5s, FR=40 rpm and FiO2 1) or HFOV (MAP 15 mmHg, FR 10 Hz, amplitude 22 and FiO2 1). The level of significance was 5%. After induction, the groups presented similar behaviors, with a decrease in the PaO2/FiO2 ratio and lung compliance, and an increase in oxygenation index (OI) and mean airway pressure (p > 0.05). At the end of experimental time, PaO2/FiO2 increased in the HFOV groups compared to the CMV groups (p < 0.05). There was a decrease in OI for HFOV groups compared to MVSG (p < 0.05), but MVPG did not differ from them (p > 0.05). There was no statistically significant difference in polymorphonuclear cell counts in bronchoalveolar lavage (BAL) in the groups with ALI. There was no difference between ALI groups regarding the TNF-alfa dosage in plasma and its gene expression in lung tissue. However, TNF-alpha measurement in BAL and in lung tissue was smaller, resembling control (p > 0.05). There was no difference in the oxidative damage assessed in the lung tissue between the groups (p > 0.05), nor between the lung regions. The histological damage score was lower in the HFOV groups, potentiated effect in the prone group when compared to the CMV groups (CG = HFPG < MVSG = MVPG), no difference in pulmonary regionalization. We conclude that, in the model of ALI induced by alveolar lavage with heated saline in rabbits: HFOV improves oxygenation if compared to CMV; PP in CMV attenuates lung inflammation, evaluated by TNF-alfa dosage in BAL and in lung tissue; ventilatory modes and positions don’t modify the oxidative stress whan evaluated by malondialdehyde method; HFOV improves histopathological lung lesion score, regardless of position, but HFOV and prone position association attenuates histopathological injury compared to protective CMV, either in the prone or supine positions. / FAPESP: 2010/06242-8

The main purpose was describe the issue of shooting success rate at high altitude (1 500 - 3 000m a. s. l.). Shooting success rate of elite racers was evaluated using analysation of results from individual races at high altitude in the period 1990/1991 - 2013/2014. High altitude had no statistical effect (p<0,05) on shooting success rate of women and men biathletes in the comparision with lowland but it had more negative effect to women shooting success rate than men shooting success rate. Ten national level biathletes were tested in three tests in lowland, four tests at high altitude and five tests in lowland after the return from high altitude. The test included rest shooting in the prone and standing positions and load shooting in both positions which took part of three kilometers running. Twelve days training camp at high altitude did not improve shooting success rate, shooting velocity and running time in the determinated heart rate. Rest shooting and shooting in the prone position did not change statistically during the whole testing period. At high altitude critical days were registered in load shooting in standing position (6th day, p<0,05) and average running time (9th day, p<0,05). Rifle manipulation and shooting are automate motions which are not influenced by high altitude. Most...

The method of directed evolution represents a new approach to generate proteins with new or altered properties. The principle of directed evolution is random mutagenesis of the coding sequence for a protein of our interest followed by selection of generated mutants for the desired property. The aim of this pilot study was to investigate the possibility of utilization of directed evolution for alteration of mouse polyomavirus original tropism and virus retargeting to a model prostate cancer cell line. To generate randomly mutated gene encoding the major capsid protein of mouse polyomavirus, which is responsible for the interaction of the virus with cellular receptor for viral cell entry, error-prone PCR and DNA shuffling methods were used. Production of viruses composed of mutant major capsid protein was ensured by Cre/loxP site-specific recombination. The thesis also dealt with the design and characterization of the system for viral mutant selection. It was found that the prostate cancer cell lines markedly vary in their ability to bind and internalize particles derived from mouse polyomavirus. This knowledge can be used for the preparation of virus-like particles for prostate cancer diagnostics in the future. The study demonstrated that the method of directed evolution can be used for production...

The main purpose was describe the issue of shooting success rate at high altitude (1 500 - 3 000m a. s. l.). Shooting success rate of elite racers was evaluated using analysation of results from individual races at high altitude in the period 1990/1991 - 2013/2014. High altitude had no statistical effect (p<0,05) on shooting success rate of women and men biathletes in the comparision with lowland but it had more negative effect to women shooting success rate than men shooting success rate. Ten national level biathletes were tested in three tests in lowland, four tests at high altitude and five tests in lowland after the return from high altitude. The test included rest shooting in the prone and standing positions and load shooting in both positions which took part of three kilometers running. Twelve days training camp at high altitude did not improve shooting success rate, shooting velocity and running time in the determinated heart rate. Rest shooting and shooting in the prone position did not change statistically during the whole testing period. At high altitude critical days were registered in load shooting in standing position (6th day, p<0,05) and average running time (9th day, p<0,05). Rifle manipulation and shooting are automate motions which are not influenced by high altitude. Most...

No description available.

Styrol-Monooxygenasen (SMOs) spielen im bakteriellen Abbau von Styrol eine wichtige Rolle. Sie epoxidieren den Kohlenwasserstoff zu (S)-Styroloxid und waren bis vor kurzem vor allem aus Gram-negativen Vertretern wie Pseudomonaden bekannt. Das Grampositive nocardioforme Bodenbakterium Rhodococcus opacus 1CP kann Styrol als Energie- und Kohlenstoffquelle nutzen und verfügt über zwei Typen von SMOs. Neben StyA2B, einer fusionierten FAD:NADH-Oxidoreduktase (StyB) und Monooxygenase (StyA2) findet sich eine weitere Monooxygenase StyA1, deren Gen direkt stromaufwärts zu styA2B lokalisiert ist. Zusätzlich zum natürlichen Fusionsprotein StyA2B gelang kürzlich die Konstruktion künstlicher Fusionen StyAL1B und StyAL2B aus Pseudomonas fluorescens ST. Um sowohl StyA1/StyA2B als auch die künstlichen Fusionen StyAL1B und StyAL2B für eine biotechnologische Anwendung nutzen zu können, wurde im Rahmen dieser Arbeit angestrebt, ihre spezifische Oxygenierungsaktivität (StyA1/StyA2B: 0,24 U/mg) mit Hilfe der error prone PCR zu erhöhen. Um Veränderungen der katalytischen Aktivität in einer großen Zahl von Mutanten schnell zu erkennen, ist ein einfacher Screeningtest erforderlich. Die Fähigkeit von SMOs zur Oxidation von Indol zu blauem Indigo bietet diese Möglichkeit. Allerdings ist hierfür die Expression löslicher Proteine eine wesentliche Voraussetzung. Versuche zur Veränderung der Gene styA2B und styA1A2B mit Hilfe eines kommerziellen error prone PCR Kits lieferten ca. 300 bis 1.200 mutmaßlich veränderte Klone, welche jedoch keinerlei Aktivität für den Indolumsatz zeigten. Als Ursache wurde eine Expression der Proteine in Form inaktiver Inclusion Bodies vermutet. Die Fusionsproteine StyAL1B und StyAL2B bilden lösliches Protein, welche Indol zum blauen Farbstoff Indigo umsetzen. Verschiedene Kultivierungsbedingungen wurden auf den Umsatz von Indol untersucht. Dabei wurde erkannt, dass die Klone sich nicht identisch bezüglich ihrer Proteinlöslichkeit verhalten. Mit Hilfe dieser Ergebnisse wurde ein Test für das Aktivitätsscreening von Styrol-Monooxygenasen auf Platte entwickelt. Die Erhöhung der NaCl-Konzentration im Medium steigerte die Indoloxidation, welche sich jedoch durch zusätzliche physiologisch Faktoren schwer beeinflussen lassen. Auch für die Fusionsproteine erfolgte die Durchführung einer error prone PCR. Der Schritt der error prone PCR stellte kein Problem dar, jedoch die Einbindung des veränderten Genfragmentes in den Vektor, beziehungsweise dessen Transformation in E. coli. Alternative Strategien, wie die Nutzung alternativer DNA Polymerasen und eines konventionellen Konzepts, bei dem veränderte Gene in geschnittene Expressionsvektoren ligiert werden, führte zu keinen detektierbaren Klonen. Die Kultivierung von identischen Klonen auf Festmedium wirkte sich aufgrund nicht näher identifizierter Einflüsse auf das Verhalten bezüglich der Indoloxidation sehr unterschiedlich aus. Um diese Einflüsse zu minimieren, erfolgte die Untersuchung des Systems in einer Flüssigkultur. Im Blickpunkt stand hierbei die Indigoproduktion von E. coli BL21 (pET_StyAL2B) die in Abhängigkeit der optischen Dichte der Kultur untersucht wurde. / Styrene monooxygenases (SMOs) play an important role in the bacterial degradation of styrene. They epoxidize the hydrocarbon highly enantioselective to (S)-styrene oxide. Most of the styrene monooxygenases known so far were identified in Gram-negative microorganisms like pseudomonads. Rhodococcus opacus 1CP, a Gram-positive nocardioform actinobacterium, which uses styrene as energy and carbon source was recently found to possess a novel type of SMO, StyA2B. This protein represents a natural fusion between an FAD:NADH oxidoreductase (StyB) and a single monooxygenase subunit (StyA2) and might act in combination with another single oxygenase StyA1 in strain 1CP. Two artificial analogs to StyA2B, designated StyAL1B and StyAL2B, were recently prepared by a fusion of styA and styB of Pseudomonas fluorescens ST and both showed oxygenating activity. For StyA1/StyA2B as well as the artificial fusion proteins StyAL1B and StyAL2B, it was tried to enhance the specific oxygenation activity in order to support their biotechnological applicability. The method of error prone PCR was used for that purpose. In order to identify favorable modifications with increased catalytic activity from a high number of mutants, an easy and simple screening test is necessary. Therefore, it is reasonable to use the ability of SMOs to oxidize indole to the blue dye indigo. However, the expression of SMOs as soluble proteins is an important requirement for any activity screening. Attempts to modify the genes styA2B and styA1/styA2B by means of a commercial error prone PCR kit yielded 300 to 1,200 potential mutants. Unfortunately, none of the obtained colonies showed any indole-oxidizing activity and the formation of insoluble inclusion bodies was assumed to be a likely explanation. In contrast to StyA2B and StyA1, recombinant expression of the artificial fused SMOs StyAL1B und StyAL2B should yield detectable amounts of active proteins. In fact, cultivation of clones expressing both types of proteins showed a blue coloration. Since the coloration of clones from one single solid medium evolved in a non-uniform manner, cultivation conditions were varied in order to identify factors which promote a more uniform tendency for indole oxidation. Although a high NaCl concentration in the medium was shown to favor indole oxidation, the latter one seems to be influenced by additional physiological factors, hardly to control. For the artificially fused proteins an error prone PCR was carried out, too. Although the initial step of mutagenic PCR was found to be successful, completing the vector system by a second ll-up PCR reaction failed. Alternative strategies like the usage of alternative DNA polymerases as well as a conventional cloning approach of various genes into a digested expression vector did not lead to detectable clones. The cultivation of identical clones on petri dishes provided no uniform tendency for indole oxidation and thus did not allow the reliable comparison of mutants in respect of their specific SMO activities. Cultivation of mutants in liquid medium should lead to more reproducible conditions and for that purpose a method was successfully established to quantify indigo formation and cell density.

Styrol-Monooxygenasen (SMOs) spielen im bakteriellen Abbau von Styrol eine wichtige Rolle. Sie epoxidieren den Kohlenwasserstoff zu (S)-Styroloxid und waren bis vor kurzem vor allem aus Gram-negativen Vertretern wie Pseudomonaden bekannt. Das Grampositive nocardioforme Bodenbakterium Rhodococcus opacus 1CP kann Styrol als Energie- und Kohlenstoffquelle nutzen und verfügt über zwei Typen von SMOs. Neben StyA2B, einer fusionierten FAD:NADH-Oxidoreduktase (StyB) und Monooxygenase (StyA2) findet sich eine weitere Monooxygenase StyA1, deren Gen direkt stromaufwärts zu styA2B lokalisiert ist. Zusätzlich zum natürlichen Fusionsprotein StyA2B gelang kürzlich die Konstruktion künstlicher Fusionen StyAL1B und StyAL2B aus Pseudomonas fluorescens ST. Um sowohl StyA1/StyA2B als auch die künstlichen Fusionen StyAL1B und StyAL2B für eine biotechnologische Anwendung nutzen zu können, wurde im Rahmen dieser Arbeit angestrebt, ihre spezifische Oxygenierungsaktivität (StyA1/StyA2B: 0,24 U/mg) mit Hilfe der error prone PCR zu erhöhen. Um Veränderungen der katalytischen Aktivität in einer großen Zahl von Mutanten schnell zu erkennen, ist ein einfacher Screeningtest erforderlich. Die Fähigkeit von SMOs zur Oxidation von Indol zu blauem Indigo bietet diese Möglichkeit. Allerdings ist hierfür die Expression löslicher Proteine eine wesentliche Voraussetzung. Versuche zur Veränderung der Gene styA2B und styA1A2B mit Hilfe eines kommerziellen error prone PCR Kits lieferten ca. 300 bis 1.200 mutmaßlich veränderte Klone, welche jedoch keinerlei Aktivität für den Indolumsatz zeigten. Als Ursache wurde eine Expression der Proteine in Form inaktiver Inclusion Bodies vermutet. Die Fusionsproteine StyAL1B und StyAL2B bilden lösliches Protein, welche Indol zum blauen Farbstoff Indigo umsetzen. Verschiedene Kultivierungsbedingungen wurden auf den Umsatz von Indol untersucht. Dabei wurde erkannt, dass die Klone sich nicht identisch bezüglich ihrer Proteinlöslichkeit verhalten. Mit Hilfe dieser Ergebnisse wurde ein Test für das Aktivitätsscreening von Styrol-Monooxygenasen auf Platte entwickelt. Die Erhöhung der NaCl-Konzentration im Medium steigerte die Indoloxidation, welche sich jedoch durch zusätzliche physiologisch Faktoren schwer beeinflussen lassen. Auch für die Fusionsproteine erfolgte die Durchführung einer error prone PCR. Der Schritt der error prone PCR stellte kein Problem dar, jedoch die Einbindung des veränderten Genfragmentes in den Vektor, beziehungsweise dessen Transformation in E. coli. Alternative Strategien, wie die Nutzung alternativer DNA Polymerasen und eines konventionellen Konzepts, bei dem veränderte Gene in geschnittene Expressionsvektoren ligiert werden, führte zu keinen detektierbaren Klonen. Die Kultivierung von identischen Klonen auf Festmedium wirkte sich aufgrund nicht näher identifizierter Einflüsse auf das Verhalten bezüglich der Indoloxidation sehr unterschiedlich aus. Um diese Einflüsse zu minimieren, erfolgte die Untersuchung des Systems in einer Flüssigkultur. Im Blickpunkt stand hierbei die Indigoproduktion von E. coli BL21 (pET_StyAL2B) die in Abhängigkeit der optischen Dichte der Kultur untersucht wurde.:Eidesstattliche Erklärung II Danksagung III Zusammenfassung IV Abstract VI Abbildungsverzeichnis XI Tabellenverzeichnis XIII Abkürzungsverzeichnis XIV 1 Einleitung 1 1.1 Styrol - ein Produkt der Industrie . . . . . . . . . . . . . . . . . . . . . . . 1 1.2 Styrol-Monooxygenasen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.2.1 Abbauwege von Styrol . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.2.2 Struktur, Vorkommen und Eigenschaften klassischer Zweikomponenten Styrol-Monooxygenasen . . . . . . . . . . . . . . . . . . . . 4 1.2.3 Das neuartige Styrol-Monooxygenase-System StyA1/StyA2B aus Rhodococcus opacus 1CP . . . . . . . . . . . . . . . . . . . . . . . . 6 1.2.4 Künstlich verlinkte SMO aus Pseudomonas uorescens ST . . . . . 7 1.2.5 Biotechnologischer Einsatz von Styrol-Monooxygenasen . . . . . . . 8 1.3 Strategien des Protein-Engineering . . . . . . . . . . . . . . . . . . . . . . 9 1.3.1 Arbeitsmethoden zur Veränderung von DNA . . . . . . . . . . . . . 9 1.3.2 Error prone PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1.4 Arbeitsziele . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 2 Material und Methoden 13 2.1 Bakterienstämme und Plasmide . . . . . . . . . . . . . . . . . . . . . . . . 13 2.2 Kultivierungsmedien und -bedingungen . . . . . . . . . . . . . . . . . . . . 14 2.2.1 Kultivierungsmedien . . . . . . . . . . . . . . . . . . . . . . . . . . 14 2.2.2 Kultivierungstemperaturen . . . . . . . . . . . . . . . . . . . . . . . 14 2.3 Polymerase-Kettenreaktion (PCR) . . . . . . . . . . . . . . . . . . . . . . 16 2.3.1 Primer und Primerdesign . . . . . . . . . . . . . . . . . . . . . . . . 16 2.3.2 Standard-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 2.4 Fehlerbehaftete Polymerase-Kettenreaktion (epPCR) . . . . . . . . . . . . 17 2.4.1 Synthese der mutagenen Megaprimer . . . . . . . . . . . . . . . . . 18 2.4.2 EZClone Reaktion . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 2.4.3 Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 2.4.4 Modi zierung des Protokolls des EZClone Reaktion Schrittes . . . . 20 2.5 Aufreinigung von PCR-Produkten aus der Lösung . . . . . . . . . . . . . . 20 2.6 TAE-Agarose-Gelelektrophorese . . . . . . . . . . . . . . . . . . . . . . . . 20 2.7 DNA-Extraktion aus Agarosegelen . . . . . . . . . . . . . . . . . . . . . . 21 2.8 Bestimmung der DNA-Konzentration . . . . . . . . . . . . . . . . . . . . . 21 2.9 Restriktionsverdau von DNA . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.10 Ligation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 2.11 Herstellung von kompetenten Zellen (E.coli DH5ff, E. coli BL21) . . . . . 23 2.11.1 Chemisch kompetente Zellen nach der CaCl2-Methode (42) . . . . . 23 2.11.2 TOP10 chemischkompetente Zellen . . . . . . . . . . . . . . . . . . 23 2.12 Transformation nach der Hitzeschock-Methode (19) . . . . . . . . . . . . . 24 2.13 Plasmidpräparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 2.14 Bestimmung der Indigobildung durch Klone mit mutmaÿlicher SMO-Aktivität 24 2.14.1 Abschätzung der Indigobildung durch Augenschein . . . . . . . . . 25 2.14.2 Quanti zierung der Indigobildung mittels UV/Vis-Spektrophotometrie 25 2.14.3 Quanti zierung der Indigobildung aus Flüssigkulturen . . . . . . . . 26 3 Ergebnisse 27 3.1 Versuche der error prone PCR von StyA2B aus Rhodococcus opacus 1CP . 27 3.1.1 Isolation von Templat-DNA und Durchführung der error prone PCR 28 3.1.2 Screening von Transformanden auf Fähigkeit zur Indol-Oxidation . 29 3.1.3 Herstellung und Aktivitätsscreening von E. coli DH5ff pET_StyA2B 30 3.2 Versuche der error prone PCR von styA1/styA2B aus Rhodococcus opacus 1CP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 3.2.1 Durchführung der error prone PCR und Aktivitätsscreening von StyA1/StyA2B in pBluescript KS(+) . . . . . . . . . . . . . . . . . 31 3.2.2 Durchführung des Aktivitätsscreening von StyA1/StyA2B in pET16bP 32 3.3 Fusionsproteine StyAL1B und StyAL2B aus Pseudomonas uorescens ST . 33 3.3.1 Optimierung der Zusammensetzung des LB-Mediums für das Aktivitätsscreenings von pET_StyAL2B in E. coli BL21 nach einer Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 3.3.2 Ein uss der Belüftung auf die Neigung von E. coli BL21 (pET_StyAL2B) Kolonien zur Oxidation von Indol . . . . . . . . . . . . . . . . . . . 38 3.3.3 Bestimmung der Indigobildung mittels UV/Vis-Spektroskopie . . . 40 3.3.4 Zeitliche Entwicklung der Indigokonzentration einer Flüssigkultur von E. coli BL21 (pET_StyAL2B) . . . . . . . . . . . . . . . . . . 42 3.3.5 Error prone PCR von pET_StyAL2B mit Gene Morph II EZ Clone Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 3.3.6 Error prone PCR nach der klassischen Methode mit pET_StyAL1B und pET_StyAL2B . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 4 Diskussion der Ergebnisse 49 4.1 Die error prone PCR als attraktive Methodik zur Optimierung von Styrol- Monooxygenasen hinsichtlich katalytischer Eigenschaften . . . . . . . . . . 49 4.2 Der Aktivitätsnachweis als mutmaÿlich limitierender Schritt in der Modi- zierung von StyA2B und StyA1/StyA2B mit Hilfe der error prone PCR . 51 4.3 Die künstlich fusionierten Styrol-Monooxygenasen StyAL2B und StyAL1B erlauben ein Aktivitätsscreening auf Platte . . . . . . . . . . . . . . . . . . 53 4.4 Die Entwicklung einer Methodik zur Quanti zierung der spezi schen Indigobildung eines Expressionsklons der Styrol-Monooxygenase StyAL2B . . . 58 4.5 Fehleranalyse zur error prone PCR . . . . . . . . . . . . . . . . . . . . . . 59 4.5.1 Fehler in der klassischen error prone PCR für pET_StyAL1B und pET_StyAL2B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Literaturverzeichnis 65 / Styrene monooxygenases (SMOs) play an important role in the bacterial degradation of styrene. They epoxidize the hydrocarbon highly enantioselective to (S)-styrene oxide. Most of the styrene monooxygenases known so far were identified in Gram-negative microorganisms like pseudomonads. Rhodococcus opacus 1CP, a Gram-positive nocardioform actinobacterium, which uses styrene as energy and carbon source was recently found to possess a novel type of SMO, StyA2B. This protein represents a natural fusion between an FAD:NADH oxidoreductase (StyB) and a single monooxygenase subunit (StyA2) and might act in combination with another single oxygenase StyA1 in strain 1CP. Two artificial analogs to StyA2B, designated StyAL1B and StyAL2B, were recently prepared by a fusion of styA and styB of Pseudomonas fluorescens ST and both showed oxygenating activity. For StyA1/StyA2B as well as the artificial fusion proteins StyAL1B and StyAL2B, it was tried to enhance the specific oxygenation activity in order to support their biotechnological applicability. The method of error prone PCR was used for that purpose. In order to identify favorable modifications with increased catalytic activity from a high number of mutants, an easy and simple screening test is necessary. Therefore, it is reasonable to use the ability of SMOs to oxidize indole to the blue dye indigo. However, the expression of SMOs as soluble proteins is an important requirement for any activity screening. Attempts to modify the genes styA2B and styA1/styA2B by means of a commercial error prone PCR kit yielded 300 to 1,200 potential mutants. Unfortunately, none of the obtained colonies showed any indole-oxidizing activity and the formation of insoluble inclusion bodies was assumed to be a likely explanation. In contrast to StyA2B and StyA1, recombinant expression of the artificial fused SMOs StyAL1B und StyAL2B should yield detectable amounts of active proteins. In fact, cultivation of clones expressing both types of proteins showed a blue coloration. Since the coloration of clones from one single solid medium evolved in a non-uniform manner, cultivation conditions were varied in order to identify factors which promote a more uniform tendency for indole oxidation. Although a high NaCl concentration in the medium was shown to favor indole oxidation, the latter one seems to be influenced by additional physiological factors, hardly to control. For the artificially fused proteins an error prone PCR was carried out, too. Although the initial step of mutagenic PCR was found to be successful, completing the vector system by a second ll-up PCR reaction failed. Alternative strategies like the usage of alternative DNA polymerases as well as a conventional cloning approach of various genes into a digested expression vector did not lead to detectable clones. The cultivation of identical clones on petri dishes provided no uniform tendency for indole oxidation and thus did not allow the reliable comparison of mutants in respect of their specific SMO activities. Cultivation of mutants in liquid medium should lead to more reproducible conditions and for that purpose a method was successfully established to quantify indigo formation and cell density.:Eidesstattliche Erklärung II Danksagung III Zusammenfassung IV Abstract VI Abbildungsverzeichnis XI Tabellenverzeichnis XIII Abkürzungsverzeichnis XIV 1 Einleitung 1 1.1 Styrol - ein Produkt der Industrie . . . . . . . . . . . . . . . . . . . . . . . 1 1.2 Styrol-Monooxygenasen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.2.1 Abbauwege von Styrol . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.2.2 Struktur, Vorkommen und Eigenschaften klassischer Zweikomponenten Styrol-Monooxygenasen . . . . . . . . . . . . . . . . . . . . 4 1.2.3 Das neuartige Styrol-Monooxygenase-System StyA1/StyA2B aus Rhodococcus opacus 1CP . . . . . . . . . . . . . . . . . . . . . . . . 6 1.2.4 Künstlich verlinkte SMO aus Pseudomonas uorescens ST . . . . . 7 1.2.5 Biotechnologischer Einsatz von Styrol-Monooxygenasen . . . . . . . 8 1.3 Strategien des Protein-Engineering . . . . . . . . . . . . . . . . . . . . . . 9 1.3.1 Arbeitsmethoden zur Veränderung von DNA . . . . . . . . . . . . . 9 1.3.2 Error prone PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1.4 Arbeitsziele . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 2 Material und Methoden 13 2.1 Bakterienstämme und Plasmide . . . . . . . . . . . . . . . . . . . . . . . . 13 2.2 Kultivierungsmedien und -bedingungen . . . . . . . . . . . . . . . . . . . . 14 2.2.1 Kultivierungsmedien . . . . . . . . . . . . . . . . . . . . . . . . . . 14 2.2.2 Kultivierungstemperaturen . . . . . . . . . . . . . . . . . . . . . . . 14 2.3 Polymerase-Kettenreaktion (PCR) . . . . . . . . . . . . . . . . . . . . . . 16 2.3.1 Primer und Primerdesign . . . . . . . . . . . . . . . . . . . . . . . . 16 2.3.2 Standard-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 2.4 Fehlerbehaftete Polymerase-Kettenreaktion (epPCR) . . . . . . . . . . . . 17 2.4.1 Synthese der mutagenen Megaprimer . . . . . . . . . . . . . . . . . 18 2.4.2 EZClone Reaktion . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 2.4.3 Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 2.4.4 Modi zierung des Protokolls des EZClone Reaktion Schrittes . . . . 20 2.5 Aufreinigung von PCR-Produkten aus der Lösung . . . . . . . . . . . . . . 20 2.6 TAE-Agarose-Gelelektrophorese . . . . . . . . . . . . . . . . . . . . . . . . 20 2.7 DNA-Extraktion aus Agarosegelen . . . . . . . . . . . . . . . . . . . . . . 21 2.8 Bestimmung der DNA-Konzentration . . . . . . . . . . . . . . . . . . . . . 21 2.9 Restriktionsverdau von DNA . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.10 Ligation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 2.11 Herstellung von kompetenten Zellen (E.coli DH5ff, E. coli BL21) . . . . . 23 2.11.1 Chemisch kompetente Zellen nach der CaCl2-Methode (42) . . . . . 23 2.11.2 TOP10 chemischkompetente Zellen . . . . . . . . . . . . . . . . . . 23 2.12 Transformation nach der Hitzeschock-Methode (19) . . . . . . . . . . . . . 24 2.13 Plasmidpräparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 2.14 Bestimmung der Indigobildung durch Klone mit mutmaÿlicher SMO-Aktivität 24 2.14.1 Abschätzung der Indigobildung durch Augenschein . . . . . . . . . 25 2.14.2 Quanti zierung der Indigobildung mittels UV/Vis-Spektrophotometrie 25 2.14.3 Quanti zierung der Indigobildung aus Flüssigkulturen . . . . . . . . 26 3 Ergebnisse 27 3.1 Versuche der error prone PCR von StyA2B aus Rhodococcus opacus 1CP . 27 3.1.1 Isolation von Templat-DNA und Durchführung der error prone PCR 28 3.1.2 Screening von Transformanden auf Fähigkeit zur Indol-Oxidation . 29 3.1.3 Herstellung und Aktivitätsscreening von E. coli DH5ff pET_StyA2B 30 3.2 Versuche der error prone PCR von styA1/styA2B aus Rhodococcus opacus 1CP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 3.2.1 Durchführung der error prone PCR und Aktivitätsscreening von StyA1/StyA2B in pBluescript KS(+) . . . . . . . . . . . . . . . . . 31 3.2.2 Durchführung des Aktivitätsscreening von StyA1/StyA2B in pET16bP 32 3.3 Fusionsproteine StyAL1B und StyAL2B aus Pseudomonas uorescens ST . 33 3.3.1 Optimierung der Zusammensetzung des LB-Mediums für das Aktivitätsscreenings von pET_StyAL2B in E. coli BL21 nach einer Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 3.3.2 Ein uss der Belüftung auf die Neigung von E. coli BL21 (pET_StyAL2B) Kolonien zur Oxidation von Indol . . . . . . . . . . . . . . . . . . . 38 3.3.3 Bestimmung der Indigobildung mittels UV/Vis-Spektroskopie . . . 40 3.3.4 Zeitliche Entwicklung der Indigokonzentration einer Flüssigkultur von E. coli BL21 (pET_StyAL2B) . . . . . . . . . . . . . . . . . . 42 3.3.5 Error prone PCR von pET_StyAL2B mit Gene Morph II EZ Clone Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 3.3.6 Error prone PCR nach der klassischen Methode mit pET_StyAL1B und pET_StyAL2B . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 4 Diskussion der Ergebnisse 49 4.1 Die error prone PCR als attraktive Methodik zur Optimierung von Styrol- Monooxygenasen hinsichtlich katalytischer Eigenschaften . . . . . . . . . . 49 4.2 Der Aktivitätsnachweis als mutmaÿlich limitierender Schritt in der Modi- zierung von StyA2B und StyA1/StyA2B mit Hilfe der error prone PCR . 51 4.3 Die künstlich fusionierten Styrol-Monooxygenasen StyAL2B und StyAL1B erlauben ein Aktivitätsscreening auf Platte . . . . . . . . . . . . . . . . . . 53 4.4 Die Entwicklung einer Methodik zur Quanti zierung der spezi schen Indigobildung eines Expressionsklons der Styrol-Monooxygenase StyAL2B . . . 58 4.5 Fehleranalyse zur error prone PCR . . . . . . . . . . . . . . . . . . . . . . 59 4.5.1 Fehler in der klassischen error prone PCR für pET_StyAL1B und pET_StyAL2B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Literaturverzeichnis 65

古今中外的任何政體都需要仰賴一個以專業為核心的文官系統，才能使主權者的各類偏好，能夠確實地轉化成政策，並且以符合經濟與效率的方式執行，方能獲致治理的績效。專業官僚系統存在的意義，就是為了使有限的公共資源，能夠符合效率及效能或其他客觀標準在社會上進行權威性的配置。 而在多元民主政體中，除了同樣需要專業之外，為了避免文官系統的專業能力被政黨或特定團體所控制，造成公共資源的配置被黨派私利過度地扭曲，因而產生了文官「中立能力」價值的呼籲。在中立能力這個帶有規範性色彩的價值引導下，常任文官的事務系統與政黨為主的政務系統間，從人事制度到政策過程，都經由某些制度設計，區隔出了一定的距離，避免了官僚系統被政黨完全掌握。 然而，中立能力這個以確保官僚系統能夠維持專業為目的的價值，在我國卻被窄化為文官與行政機關不應涉入選舉與政黨活動的「行政中立」概念，而忽略了這些制度設計背後更重要的目的，討論的範圍也被限縮在行政中立法上，而沒能處理到政策過程以及官僚系統在組織管理與運作的部分。 本研究有兩個主要的目的，首先，在理論與概念的層次上，經由檢閱歐美民主國家對於中立能力概念的理論與實證研究，嘗試為國內對中立能力此一尚屬陌生卻又影響深遠的概念重新聚焦深化。其次，在實證研究的資料累積上，透過「易淹水地區水患治理計畫」這個政策個案，深度訪談水利署與地方政府水利單位官員，輔以官方報告、報章雜誌等次級資料的比對，探索在真實複雜的政策個案中，文官與行政組織能否實踐以專業為核心的中立能力價值，又是什麼樣的因素或是制度設計能夠確保其維持中立。 本研究最終發現，在理論與概念的層次上，中立能力價值又能夠區分為兩項內涵，分別是居於核心的專業主義以及作為指導與外界互動界面上的公正性。而在實證的政策個案研究上，易淹水地區水患治理計畫經由「以流域為整體單位的規劃報告」，嚴格要求工程的施作必須符合規劃中的整體性與急迫性，並且建立起從中央到地方對於工程施作的三級審核制度，使得治水工程的預算經費配置大致符合了專業的客觀標準，並且在近年的歷次颱風水患中發揮效用。在此個案中，專業規劃的報告與審查制度確保了中立能力的實踐與政策的果效。