Submitted by Eliézer Fernando de Oliveira null (eliezer@fc.unesp.br) on 2016-12-01T22:42:08Z No. of bitstreams: 1 tese-oliveiraef-final-2016.pdf: 4897359 bytes, checksum: e91f8fa951ca10c244974954f09cb53b (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-12-05T13:21:08Z (GMT) No. of bitstreams: 1 oliveira_ef_dr_bauru.pdf: 4897359 bytes, checksum: e91f8fa951ca10c244974954f09cb53b (MD5) / Made available in DSpace on 2016-12-05T13:21:08Z (GMT). No. of bitstreams: 1 oliveira_ef_dr_bauru.pdf: 4897359 bytes, checksum: e91f8fa951ca10c244974954f09cb53b (MD5) Previous issue date: 2016-11-28 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Materiais orgânicos vêm sendo utilizados em diversos tipos de dispositivos eletrônicos e optoeletrônicos, sendo um dos destaques seu uso em células solares. Atualmente, células solares que utilizam materiais orgânicos na camada ativa já atingem eficiências em torno de 12% na conversão da energia solar em elétrica. Apesar destes materiais apresentarem vantagens em relação ao custo do produto final e processamento comparado aos inorgânicos, a comunidade científica ainda se depara com problemas relacionados às propriedades intrínsecas, como por exemplo, baixa estabilidade à oxidação e energias de bandgap elevadas; deste modo, ainda é viável encontrar novos materiais orgânicos que superem tais problemas. Este trabalho teve como finalidade a busca de novos materiais orgânicos, mais especificamente polímeros conjugados, para aplicações em camada ativa de células solares utilizando métodos teóricos de modelagem de materiais em níveis de teoria semi-empírico e DFT. A primeira parte deste trabalho focou no estudo de novos copolímeros de comonômeros similares e novos homopolímeros derivados de P3HT. Aqui foi possível verificar que, através de substituições químicas realizadas no P3HT, pode-se encontrar novos polímeros com valores mais apropriados para as energias dos orbitais moleculares de fronteira e maiores mobilidades de carga, propriedades que estão intimamente relacionadas à eficiência e à estabilidade que a célula solar pode atingir. Um dos novos polímeros que apresentou potencial para aplicação em camadas ativas de células solares foi o P3HT fluorado, que posteriormente foi confirmado através de trabalhos experimentais. Para os copolímeros de comonômeros similares, verificou-se que as propriedades eletrônicas e ópticas destes materiais apresentam uma dependência linear com as mesmas propriedades e a proporção de monômeros dos homopolímeros que foram utilizados como comonômeros na construção do copolímero; o destaque maior é que através desta relação linear pode-se projetar novos copolímeros de comonômeros similares antes de uma possível síntese. A segunda parte deste trabalho avaliou as metodologias de modelagem de materiais orgânicos baseadas na (TD)DFT que seriam viáveis para a correta descrição teórica das propriedades ópticas. Verificou-se que uma confiável previsão da energia de transição vertical pode ser obtida por meio do funcional M06HF, adicionando um fator de correção de -0,75 eV nas energias obtidas com ele. Em relação à absorção óptica do estado excitado, o funcional B3LYP não prevê corretamente as energias de transição entre os estados excitados, ao passo que resultados mais confiáveis podem ser obtidos com os funcionais BHLYP e CAM-B3LYP comparado aos dados experimentais disponíveis. / Organic materials have been used in various types of electronic and optoelectronic devices, with prominent application in solar cells. Solar cells in which the active layers are composed of organic materials already reach efficiencies of around 12% in the conversion of solar energy into electricity. Although these materials have advantages in relation to the final cost and processing compared to the inorganics, the scientific community still faces problems related to intrinsic properties, such as low oxidation stability and high bandgap energy; thus, it is desirable to find new organic materials that overcome these problems. This work aimed at the search for new organic materials – specifically conjugated polymers – for applications in active layers of solar cells employing theoretical methods of material modeling of semi-empirical and DFT theory levels. The first part of this work focuses on the study of new copolymers of similar comonomers and new P3HT derivatives. Here it was observed that, through chemical substitutions made in P3HT, one may find new polymers with appropriate values for the energies of the frontier molecular orbitals and increased charge mobility, properties that are closely related to the efficiency and stability that the solar cell can achieve. One of the novel polymers found which have potential for use in active layers was the fluorinated P3HT, later confirmed by experimental works. For copolymers of similar comonomers, it was found that the electronic and optical properties of these materials exhibit a linear dependence with the same properties and the monomers proportion of the homopolymers that were used as comonomers in the construction of the copolymer; most important is that through this linear relationship one may design new copolymers of similar comonomers before a possible synthesis. The second part of this study evaluated the methodologies for modeling of organic materials based on (TD)DFT that would be viable for a proper theoretical description of the optical properties. It was found that a reliable prediction of the vertical transition energy from the ground state of conjugated polymers can be obtained with the M06HF functional adding a correction factor of -0.75 eV in its obtained energies. Regarding the excited state optical absorption, the B3LYP functional do not correctly predicts the transition energy between the excited states, while more reliable results can be obtained with BHLYP and CAM-B3LYP functionals compared to the available experimental data. / FAPESP: 2012/21983-0

Made available in DSpace on 2017-07-10T17:40:56Z (GMT). No. of bitstreams: 1 Daniel_Schwantes.pdf: 5686979 bytes, checksum: e1296696716d8829be7043393332c024 (MD5) Previous issue date: 2016-08-05 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This research relates the development and use of modified adsorbents from agroindustrial residues. The following solid agroindustrial residues were obtained: pine bark (Pinus elliottii), solid residue from the manufacture of wood; Crambe cake (Crambe abyssinica Hochst), solid residue from oil extraction (biodiesel agroindustry); and cassava peels (Manihot esculenta Crantz), a byproduct of cassava industrialization. Biosorbents P. in natura, C. in natura and M. in natura (natural adsorbents or biosorbents) were produced from these agroindustrial residues, which had no or low market value, from which were produced the modified adsorbents, by washing the biomass with solutions of H2O2, H2SO4 and NaOH 0.1 mol L-1, resulting the modified adsorbents of pinus P. H2O2, P. H2SO4 and P. NaOH; crambe C. H2O2, C. H2SO4 and C. NaOH, and cassava M. H2O2, M. H2SO4, M. NaOH. In the first step of the study of these adsorbents (in natura and modified) the adsorbent materials were characterized, namely: chemical composition of the adsorbents, pHPCZ, infrared spectra (FT-IR), scanning electron microscopy (SEM), thermogravimetry (TG and DTG), BET and BJH isotherms (porosity and surface area). After the characterization of the adsorbent materials, adsorption studies were carried out for the evalution of Cd(II), Pb(II) and Cr(III) removal, which aimed to evaluate the performance of the materials on the influence of the pH of contaminant solution (ideal adsorption conditions), ie, the adsorption of metal ions as a function of increasing masses and the pH of the medium; the influence of adsorption time (adsorption kinetics), the influence of increasing concentrations of metal ions and isotherm linearization (adsorption equilibrium studies), the effect of temperature on the sorption process (adsorption studies) as well as the reuse of these adsorbent materials by acid elution (desorption studies). The scanning electron microscopes indicated changes in the adsorbents surface; the pHPZC of the modified adsorbents was distinct from the biosorbents. In addition, changes were also observed in the bands of the functional groups in the infrared spectrum, with new functional groups observed, such data indicate that modifications occurred to the adsorbents as a function of the chemical treatment applied. The results showed that, in general, the modified adsorbents present higher adsorption capacity of Cd(II), Pb(II) and Cr(III) metal ions than in natura materials. The modified adsorbents based on pinus, crambe and cassava did not show significant variation in relation to the studied pH range, which is a great advantage, because the adsorbents developed adapt to a wide pH range of contaminated water. An average time of 40 minutes was found for adsorption equilibrium, indicating that the sorption process is fast and occurs in the first few moments. The adsorption of the toxic ions occurs in mono and multilayer, in agreement with the good adjustments observed for Langmuir and Freundlich. In general, the adsorbents presented good reuse capacities, except when used for Cr(III) adsorption, because in this case it is difficult to reuse the adsorbents due to the low desorption rates of this metal, possibly due to chemisorption. Based on the results obtained, it can be concluded that the adsorbents of pinus, crambe and cassava present themselves as excellent, renewable, high availability and low cost, being an attractive alternative for the industry, through its use in advanced treatment systems, for the removal of metals such as Cd(II), Pb(II) and Cr(III) / Esta pesquisa relaciona o desenvolvimento e uso de adsorventes modificados a partir de resíduos agroindustriais. Para tal, foram obtidos os seguintes resíduos sólidos agroindustriais: cascas de pinus (Pinus elliottii), resíduo sólido da fabricação de madeira; torta de crambe (Crambe abyssinica Hochst), resíduo sólido da extração do óleo (agroindústria do biodiesel); e cascas de mandioca (Manihot esculenta Crantz), subproduto da industrialização da mandioca. A partir destes resíduos agroindustriais, que apresentam nenhum ou baixo valor de mercado, foram produzidos os biossorventes P. in natura, C. in natura e M. in natura e (adsorventes naturais ou biossorventes), dos quais foram produzidos os adsorventes modificados, por meio da lavagem com soluções de H2O2, H2SO4 e NaOH 0,1 mol L-1, dando origem aos adsorventes modificados de pinus P. H2O2, P. H2SO4 e P. NaOH; de crambe C. H2O2, C. H2SO4 e C. NaOH, e de mandioca M. H2O2, M. H2SO4, M. NaOH. Na primeira etapa do estudo destes adsorventes (in natura e modificados), realizou-se a caracterização dos materiais, a citar: composição química dos adsorventes, pHPCZ, espectros de infravermelho (FT-IR), microscopia eletrônica de varredura (MEV), termogravimetria (TG e DTG) e isotermas de BET e BJH (porosidade e área superficial). Após a caracterização dos materiais adsorventes, foram conduzidos estudos de adsorção para os metais Cd(II), Pb(II) e Cr(III), que visavam avaliar a performance dos materiais quanto a influência do pH da solução contaminante (condições ideais de adsorção), ou seja, a adsorção de íons metálicos em função de massas crescentes e do pH do meio; o efeito do tempo no processo de adsorção (cinética de adsorção), a influência de concentrações crescentes de íons metálicos e linearização das isotermas (estudos de equilíbrio de adsorção), o efeito da temperatura no processo sortivo (estudos de termodinâmica de adsorção), bem como o reuso destes materiais adsorventes por meio de eluição ácida (estudos de dessorção). As microscopias eletrônicas de varredura indicaram a ocorrência de mudanças na superfície dos adsorventes, o pHPCZ dos adsorventes modificados foi distinto dos biossorventes, além disso, também foram observadas alterações quanto a amplitude das bandas dos grupos funcionais no espectro de infravermelho, com novos grupos funcionais observados, tais dados sugerem a ocorrência de modificações aos adsorventes. Os resultados obtidos demonstram que, no geral, os adsorventes modificados apresentam maior capacidade de adsorção de íons metálicos Cd(II), Pb(II) e Cr(III) que os materiais in natura. Os adsorventes a base de pinus, crambe e mandioca não apresentaram variação significativa com relação à faixa de pH estudada, sendo esta uma grande vantagem, pois os adsorventes desenvolvidos se adaptam a uma ampla faixa de pH de águas contaminadas. Foi encontrado um tempo médio de 40 minutos para ocorrência do equilíbrio de adsorção, indicando que o processo sortivo é rápido e ocorre nos primeiros instantes. A adsorção dos íons ocorre em mono e multicamadas, em concordância com os bons ajustes observados para Langmuir e Freundlich. Em geral, os adsorventes apresentaram boa capacidade de reuso, exceto quando utilizados para adsorção de Cr(III), pois neste caso ocorre dificuldade de reuso dos adsorventes pela baixa taxa de dessorção deste metal, indicando possível quimissorção. Pelos resultados obtidos conclui-se que os adsorventes modificados de pinus, crambe e mandioca apresentam-se como excelentes, renováveis, de alta disponibilidade e baixo custo, sendo uma alternativa atraente para a indústria, mediante sua utilização em sistemas de tratamento avançados, para a remoção de metais como Cd(II), Pb(II) e Cr(III)

Orientadores: Sergio Marangoni, Luís Alberto Ponce Soto / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T23:14:07Z (GMT). No. of bitstreams: 1 Vega_SalomonHuancahuire_D.pdf: 1506157 bytes, checksum: 874bfba78d521b4e037ba15f754d80b7 (MD5) Previous issue date: 2012 / Resumo: No presente trabalho, a partir do veneno de Porthidium hyoprora, foram purificadas duas fosfolipases A2 (PLA2) miotóxicas, denominadas PhTX-I e PhTX-II, determinadas suas sequências primárias e caracterizadas bioquímica e farmacologicamente. Ambas as proteínas foram purificadas em uma única etapa cromatográfica (HPLC-FR), sendo obtida homogênea e com alto grau de pureza, confirmado por SDS-PAGE, análise de aminoácidos e espectrometria de massas. PhTX-I e PhTX-II estão constituídas de uma única cadeia polipeptídica e possuem massas moleculares de 14.249 e 14.149 Da, respectivamente. A sequência de aminoácidos foi determinada via espectrometria de massas ESI-MS/MS, pertencendo à família das PLA2 D49. A estrutura secundária predominante destas proteínas está constituída de ?-hélices. PhTX-I e PhTX-II induziram atividade paralisante neuromuscular ex vivo na preparação de biventer cervicis de pintainho e esta atividade foi similar ao efeito de outras PLA2 de veneno botrópico. In vivo, tanto PhTX-I quanto PhTX-II, induziram elevada miotoxicidade local e atividade edematogênica. Adicionalmente, PhTX-I induz liberação de IL-6 após injeção intramuscular em camundongos. In vitro, PhTX-I e PhTX-II foram citotóxicas de maneira dose-dependente para as linhagens celulares de fibroblastos NIH-3T3 e NG97 derivada de astrocitoma humano grau III. Adicionalmente PhTX-I foi citotóxica sobre cultura celular de miotubos C2C12, entretanto teve pouco efeito citolítico sobre mioblastos de músculo esquelético. Foram feitas modificações químicas em resíduos de aminoácidos específicos da PhTX-I e avaliadas suas atividades farmacológicas após as modificações. Foram modificados um resíduo de His e Trp, quatro de Tyr e sete de Lys. A análise destas proteínas por dicroísmo circular demonstrou que a estrutura secundária das proteínas modificadas não foi alterada significantemente. Avaliação das atividades biológicas indicam um papel crítico desempenhado pelos aminoácidos Lys e Tyr na miotoxicidade, efeito paralisante neuromuscular e principalmente na citotoxicidade induzida por PhTX-I. A atividade catalítica da PhTX-I é relevante para os efeitos edematogênico, paralisante neuromuscular e miotóxico, mas não para sua atividade citotóxica, pois este efeito foi completamente independente da atividade catalítica de PhTX-I, evidenciando a existência de regiões moleculares, diferentes a do sítio catalítico, as quais podem ser responsáveis por pelo menos algumas das propriedades farmacológicas da PhTX-I. Podemos concluir, que apesar de ser demonstrada uma dissociação parcial, tanto o sítio catalítico como os sítios farmacológicos hipotéticos são relevantes para o perfil farmacológico de PhTX-I / Abstract: In this work, were purified two myotoxics phospholipases A2 (PLA2), PhTX-I and PhTX-II, from P. hyoprora snake venom, determined their primary sequences and characterized their pharmacological and biochemical activities. These proteins were purified in a single chromatographic step (FR-HPLC) and obtained with homogeneous and high purity, which was confirmed by SDS-PAGE, amino acid analysis and mass spectrometry. PhTX-I and PhTX-II are constituted of a single polipetide chain and have molecular mass of 14.249 and 14.149 Da, respectively. The amino acid sequence was determined by ESI-MS/MS mass spectrometry; belong to PLA2 D49 family. The predominant secondary structure of these proteins consists of ?-helix. PhTX-I and PhTX-II induced neuromuscular paralyzing effect ex vivo in young chick biventer cervicis preparations, this activity was similar to other PLA2 of snake venom. In vivo, PhTX-I and PhTX -II induced local myotoxicity and edema-forming activity, in addition, PhTX-I induces release of IL-6 after intramuscular injection in mice. In vitro, PhTX-I and PhTX -II were cytotoxic to the fibroblast cell line NIH-3T3 and NG97 derived from grade III human astrocytoma. Additionally PhTX-I induced low cytotoxicity in skeletal muscle myoblasts, however was able to lyse myotubes. Were made modifications chemical in PhTX-I specific amino acid residues and evaluated the pharmacological activities after modifications. Was modified one His and Trp, four Tyr and seven Lys residues? Analysis by circular dichroism demonstrated that secondary structure of protein remains practically unchanged. Evaluation of biological activities indicate a critical role of Lys and Tyr amino acid at myotoxicity, neuromuscular paralyzing effect, and especially cytotoxicity induced by PhTX-I. The catalytic activity of PhTX-I is relevant to the edematogenic, neuromuscular paralyzing and myotoxic effects, but not for their cytotoxic activity, since this effect is completely independent of the catalytic activity of PhTX-I, demonstrating the existence of molecular regions, different from the catalytic site, which may be responsible for at least some of the pharmacological properties of PhTX-I. We conclude that both the catalytic site and the hypothetical pharmacological site(s) are apparently relevant for the pharmacological profile of PhTX-I, although a partial dissociation between these activities has been demonstrated / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

L’objectif de ces travaux consiste à développer de nouvelles briques de terre cuite à base d’agents porogènes biosourcés dans le but d’obtenir des matériaux à haute performance thermique. Les matières premières, argile et biomasse, ont d’abord été caractérisées de manière individuelle. L’utilisation de co-produits agricoles (paille de blé, tourteau de tournesol et farine de noyaux d’olive) en tant qu’agents porogènes pour l’obtention de briques de terre cuite porosées a ensuite été étudiée. L’intérêt d’ajouter des éléments biosourcés entraînant une augmentation de la porosité, une augmentation de la résistance thermique malgré une diminution des propriétés mécaniques a alors été démontré. Toutefois, des problèmes de plasticité causant des soucis de mise en forme au niveau des échantillons ont été identifiés. Après avoir optimisé le taux de sable, l’impact de la granulométrie des matières végétales ainsi que leur taux d’incorporation ont été évalués. Une porosité maximale de 34,4 %, constituée de macropores, a été obtenue dans les conditions expérimentales optimales conduisant à une conductivité thermique de 0,45 W/m.K, soit une diminution de 15 % par rapport à un échantillon de référence sans matière végétale. Afin de continuer à améliorer les propriétés du matériau argileux obtenu, il a ensuite été envisagé de créer une microporosité en modifiant chimiquement les matières végétales qui seraient ensuite dégradées lors de la cuisson. Pour cela, les matières végétales ont été modifiées soit par imprégnation directe de carbonates soit par greffage de nouvelles fonctions carbonates ou esters. Les matières ainsi modifiées ont ensuite été incorporées dans les formulation de terre cuite et les propriétés des matériaux obtenus ont été mesurées (physiques, mécanique et thermique). Enfin, l’impact environnemental potentiel des échantillons contenant des additifs modifiés a été étudié à travers une Analyse de Cycle de Vie. Les différents scénarios, simples (avec un seul agent porogène) ou combinés (avec un agent végétal et un agent de synthèse) ont été comparés avec la méthode ReCiPe. Les étapes présentant les impacts les plus importants ont été mises en exergue. Il a également été démontré que le choix de l’unité fonctionnelle pouvait modifier radicalement les conclusions de l’étude. / The objective of this work is to develop new clay bricks with biobased pore-forming agents in order to obtain materials with high thermal performance. The raw materials, clay and biomass, were first characterized individually. The use of agricultural by-products (wheat straw, sunflower seed cake and olive stone flour) as pore-forming agents for the production of porous clay bricks was then studied. The benefits of the addition of biobased components that increase the porosity and the thermal resistance was demonstrated even if it results in a decrease of mechanical properties. However, problems of plasticity causing trouble during the extrusion of samples were identified. After optimizing the sand rate, the impact of the particle size of the biomass and the rate of incorporation were assessed. A maximum porosity of 34.4%, made up of macropores was obtained under optimal experimental conditions, leading to a thermal conductivity of 0.45 W/m.K, representing a decrease of 15 % compared to the reference brick without additive. In order to improve the properties of the resulting material, it was considered to create microporosity, thanks to the chemical modification of the vegetable materials that will be degraded during the firing process. For this, the agricultural by-products were modified either by direct impregnation of carbonates or by grafting of new chemical moieties (carbonate or ester). These modified matters were then incorporated into the clay formulation and the properties of the obtained bricks were measured (physical, mechanical and thermal ones). Finally, the potential environmental impact of the modified samples containing additives was studied through a Life Cycle Assessment. Different scenarios, simple (with one pore-forming agent) or combined (with a crude vegetable agent and a synthesized one) were compared with the ReCiPe method. The steps from the process with the most significant impacts were highlighted. It was also shown that the choice of the functional unit could radically alter the conclusions of the study.

CRISPR/Cas9 induced DNA breaks can be precisely repaired by cellular homology-directed repair (HDR) pathways using exogenously provided template DNA (donor). However, the full potential of precision editing is hindered in many model systems by low cutting efficiencies, low HDR efficiencies and, cytotoxicity related to Cas9 and donor DNA. In this thesis, I address these challenges and present methods that we developed to increase HDR efficiencies in multiple model organisms. In Caenorhabditis elegans, we show that by reducing toxicity high editing efficiencies can be achieved with single stranded oligonucleotide (ssODN) donors. We demonstrate that melting dsDNA donors dramatically improves the knock-in efficiencies of longer (1kb) edits. In addition, we describe 5′-terminal modifications to the donor molecules that further increase the frequency of precision editing. With our methodology a single optimally injected animal can yield more than 100 Green Fluorescent Protein (GFP) positive progeny, dramatically enhancing efficiency of genome editing. Next, we demonstrate the generality of 5′ modified donors by extending our studies to human cell cultures and mice zygotes. In mammalian models, 2′OMe-RNA modifications consistently increase HDR efficiencies by several fold over unmodified donors. Furthermore, end-modified donors exhibited a striking reduction in end-joining reactions including reduced concatemer formation and reduced direct ligation into the host genome. Our study demonstrates that HDR can be improved without inhibiting competing end-joining pathways and provides a platform to identify new chemical modifications that could further increase the potency and efficacy of precision genome editing.

Die hier vorliegende Arbeit beschäftigt sich mit Eignung bakterieller Hüllproteine als Bindungsmatrix für die Kopplung funktionaler Moleküle mit dem Ziel, sensorische Schichten zu erzeugen. Bakterielle Hüllproteine sind biologische SAMs, anderen Oberfläche sich modifizierbare COOH-, NH2- und OH-Gruppen befinden. Die Ausbildung polymerer Strukturen erfolgt dabei in wässrigen Systemen und auf Oberflächen. Im Zuge der boomenden Entwicklung von Biosensoren werden insbesondere Biotemplate gesucht, die zwischen biologischer Komponente und Sensoroberfläche vermitteln. Bakterielle Hüllproteine stellen eine solche Zwischenschicht dar. Als Anwendungsbeispiel wurden die Proteine daher mit einem FRET-Paar und Thrombin und Kanamycin-Aptameren modifiziert. Hierbei wurden das FRET-Paar H488 und H555 an die bakteriellen Hüllproteine der beiden Haldenisolate A12 und B53 mittels EDC mit einer Modifizierungsrate von 0,54 molFarbstoff/molProtein kovalent gebunden. Bei der vorhandenen p4-Symmetrie bedeutet dies, dass ein FRET-Paar pro Einheitszelle vorhanden war. Der Nachweis eines Energietransfers zwischen den beiden am Protein gebundenen Fluoreszenzfarbstoffen H488 und H555 erfolgte mittels statischer und zeitaufgelöster Fluoreszenzmessung. Die Ergebnisse zeigten, dass ein Energietransfer nur möglich war, wenn die Proteine in polymerer Form vorlagen, unabhängig davon, ob sich die Proteine immobilisiert an einer Oberfläche oder in wässriger Lösung befanden. Mittels Variieren des Donor-Akzeptor-Verhältnisses konnte ein maximaler Energietransfer von 40 % generiert werden, wenn das Verhältnis der Fluoreszenzfarbstoffe von Donor und Akzeptor 4 betrug. Die Fluoreszenzintensität der Fluorophore wurde durch die Bindung an die Proteine nicht verringert oder gelöscht. Dies legt nahe, dass die Farbstoffe in den hydrophoben Poren immobilisiert wurden und die Poren die Fluoreszenzfarbstoffe schützen. Um weitere Aussagen über die Lage der gebundenen Fluoreszenzfarbstoffe zu erhalten, wurden die bakteriellen Hüllproteine der Stämme A12 und B53 enzymatisch verdaut und die Fragmente mittels SEC und SDS-PAGE untersucht. Dabei zeigten sich je nach Enzym und Protein unterschiedliche Bandenmuster bezüglich modifizierter und nativer Hüllproteine. Dies belegt, dass die Fluoreszenzfarbstoffe an NH2-und COOH-Gruppen der Proteine gebunden wurden und so teilweise den enzymatischen Verdau hinderten. Die SEC deutet an, dass die Fluoreszenzfarbstoffe an verschiedenen Stellen am Protein gebunden wurden. In einem zweiten Beispiel wurde das bakterielle Hüllprotein von A12 mit einem Aptamer modifiziert. Aptamere sind kurze einzelsträngige Oligonukleotide, die u.a. mittels ihrer ausgebildeten 3D-Struktur spezifisch Zielstrukturen reversibel binden können. Die hier verwendeten Aptamere binden spezifisch Thrombin und Kanamycin. Die Aptamere wurden mit Hilfe einer der beiden Vernetzer PMPI oder Sulfo-SMCC an die bakteriellen Hüllproteine kovalent gebunden. Nach dem Modifizieren der Proteine wurden diese auf entsprechenden Sensorchips immobilisiert und die Aktivität des gekoppelten Aptamers mittels Affinitätsmessungen, SPR-Spektroskopie und QCM-D-Messungen analysiert. Die Funktion des gebundenen Thrombinaptamers konnte mittels Affinitätsmessungen und QCM-D nachgewiesen werden und entspricht in beiden Fällen einer Bindung von 2 nmol Thrombin pro Quadratzentimeter. Die Funktionalität des Kanamycinaptamers sollte mittels SPR bestimmt werden, jedoch konnte keine Funktionalität des gekoppelten Kanamycinaptamers nachgewiesen werden. Alle Messungen bestätigten jedoch, dass die Bindungsmatrix aus bakteriellen Hüllproteinen keinerlei oder nur ein sehr geringes Hintergrundsignal liefert. Werden nun beide Komponenten, FRET-Paar und Aptamere, an das Protein gebunden, ist es möglich, eine sensorische Schicht zu erzeugen. Die Zielstruktur, welche detektiert werden soll, wird an das Aptamer gebunden und so in räumliche Nähe zur Sensorfläche gebracht. Stell die Zielstruktur einen Fluoreszenzlöscher dar, so wird der Energietransfer durch die räumliche Nähe des Fluoreszenzlöscher gestört. Die Detektion des Zielmoleküls erfolgt nun über die Änderung von Fluoreszenzintensitäten. Die hier vorgelegte Arbeit soll einen Grundstein legen für die Entwicklung eines solchen Sensors und insbesondere die Detektion eines Energietransfers optimieren und Schwachstellen in der Detektion nachweisen. Die systematische Untersuchung der Fluoreszenzfarbstoffe auf dem Protein ermöglichen es, in zukünftigen Arbeiten einen FRET zweifelsfrei zu detektieren. Die Modifizierung von bakteriellen Hüllproteinen von A12 mit Aptameren und die Detektion der Funktionalität der Aptamere mittels verschiedener Methoden zeigte auf, dass die bakteriellen Hüllproteine als universelle Bindungsmatrix für sensorische Moleküle dienen können, bei denen Affinitätsmessungen, SPR- oder QCM-D-Messungen genutzt werden. Besonders hervorzuheben ist, dass bakterielle Hüllproteine nahezu kein Hintergrundsignal liefern und aufgrund ihrer dünnen Monolage von etwa 6 - 9 nm die Sensitivität der Messungen nur gering beeinträchtigen.

Bile acids in the small intestines of animals serve to breakdown fats and fatsoluble vitamins. Most of the bile acids are reabsorbed into the enterohepatic circulation, but approximately five percent of these bile acids pass into the large intestine. These bile acids are swiftly deconjugated by the bacterial population, and then subjected to further intestinal bacterial chemical modifications. The most significant of these modifications are 7α-dehydroxylations which form secondary bile acids (deoxycholate and lithocholate). Much research has illuminated the 7α-dehydroxylation pathway: of particular interest is the bile acid inducible operon, for which Clostridium scindens VPI 12708 serves as the model organism. There is a lack of knowledge on how this operon is regulated, so the goal of this project was to create a genetic construct consisting of upstream regulatory elements, a bile acid inducible promoter, and a ϐ- glucuronidase reporter. Cloning strategies utilized PCR to amplify desired DNA fragments and sewing methodology to combine DNA fragments. DNA fragments were ligated into plasmids and transformed into competent E. coli. Transformants were evaluated for the desired reporter gene fusion by blue/white screening, additional PCR, and/or restriction digestion. The bile acid inducible promoter was successfully amplified, and the upstream sequence and uidA (ϐ- glucuronidase) reporter was demonstrated. However, no E. colitransformants were demonstrated to possess the baiP-uidA gene fusion. The project strategy is plausible and data regarding the bile acid inducible promoter are greatly needed.