One of the most prominent factors affecting the performance and longevity of risers is vortex induced vibration (VIV), which can cause severe fatigue damage, especially in risers used in deep waters. The available approaches for analyzing VIV induced fatigue in risers mainly focus on the VIV aspect of the problem; indeed less attention has been paid on the effect of VIV on a riser’s fatigue life and in prediction of fatigue life using various models. This dissertation first demonstrates how one can characterize fatigue of pipes and risers using an equivalent plate specimen as opposed to using a pipe specimen, thereby simplifying the task, yet obtaining good accuracy. Actual variable amplitude loadings (VAL) are used to study the fatigue crack growth in risers’ material with a focus on the various influencing parameters. Extensive experimental investigations are performed, followed by analytical and computational nonlinear finite element analyses. It is shown that the higher harmonics do cause significant fatigue damage, thus their influence should not be ignored. The influence of load interaction effects is also investigated, focusing on the fatigue crack growth retardation effects due to tension overloads, as well as the acceleration effects due to compression underloads. The crack closure concept is then used to explore into both the fatigue retardation and acceleration effects within a VAL scenario. An effective method for calculation of the stress intensity factor is proposed, which considers only the tensile portion of the stress range, while proposing another effective approach for accounting for the influence of compressive stress cycles. Moreover, a two-parameter approach is used in this dissertation, relating the fatigue crack growth rate (FCGR) to the crack tip opening displacement (CTOD). It is shown that the CTOD provides adequate information for calculating the FCGR under VAL, and it can be effectively used to account for the influence of the compressive stress cycles. The experimental investigation also considers the retardation effect resulting from the applied peak tensile overload cycles (TOLC) and the influence of various so-called “clipping” levels, demonstrating the significant influence of the TOLC on crack growth retardation in VAL.

The aim of this work is to analyse the methods of valuation in accounting in accordance with Czech Accounting Legislation and International Financial Reporting Standards. This thesis compares different approaches on the example of the specific accounting entity. The company is called E.ON Czech Republic, s. r. o. The theoretical part of the thesis describes accounting systems and defines some of the international standards. The practical part of the thesis characterizes the accounting entity and describes the found differences. The next point is the subsequent comparison and evaluation.

O crescimento populacional acelerado é o grande motivador para o desenvolvimento do setor da construção civil e o aumento na demanda por água potável, tendo como consequência, um aumento gradativo na geração de resíduos sólidos. Dessa forma, este trabalho foi realizado com o intuito de reciclar resíduos industriais e municipais incorporando-os em materiais para a construção civil. O compósito produzido a partir de lodo de estação de tratamento de água e lama de polimento de mármore, aplicando resíduo de produção de cal como ligante, foi avaliado quanto ao seu desempenho mecânico e sua estrutura morfológica. As matérias-primas foram caracterizadas quanto sua composição química, mineralógica,morfológica, granulométrica e, também, o teor de umidade. Com os materiais caracterizados, foram desenvolvidas nove composições variando-se o teor de lodo de estação de tratamento de água entre 25 e 50%, o teor de lama de polimento de mármore entre 35 e 50% e o resíduo de produção de cal entre 10 e 30%. Os compósitos foram submetidos a ensaios de resistência mecânica, absorção de água, análise química, mineralógica e morfológica. Os materiais desenvolvidos apresentaram, no 3° dia de cura, valor de resistência mecânica máxima de 4,65 MPa, no 7° dia 6,36 MPa, no 14° dia 6,74 MPa, no 28° dia 5,98 MPa, no 60° dia 8,52 MPa, no 90° dia 11,75 MPa e no 180° dia 12,06 MPa. Os valores de absorção de água aos 28 dias de cura variaram de 16,27 a 26,32% e aos 90 dias, de 13,57 a 23,56%. / The rapid population growth is the great motivator for the development of the construction industry and the increased demand for drinking water, resulting in a gradual increase in the generation of solid waste. Thus, this work was carried out in order to recycle industrial and municipal wastes incorporating them into materials for civil construction. The composite produced from water treatment sludge and marble polishing mud, applying lime production waste as a binder, was evaluated for its mechanical performance and its morphological structure. The raw materials were characterized for their chemical composition, mineralogy, morphology, particle size and also the moisture content. With the featured materials nine compositions have been developed varying the content of the water treatment sludge between 25 to 50%, marble polishing mud between 35 to 50% and the lime production waste between 10 to 30%. The composites were subjected to mechanical strength tests, water absorption, chemical and mineralogical composition and morphology. The developed materials presented, on the 3rd day of hydration, maximum strength value of 4.65 MPa, the 7th day 6.36 MPa, on the 14th day 6.74 MPa, the 28th day 5.98 MPa, on the 60th day 8.52 MPa at 90th day 11.75 MPa and 180th day 12.06 MPa. The water absorption values after 28 days of hydration ranged from 16.27% to 26.32% and after 90 days, from 13.57% to 23.56%.

Os resíduos decorrentes da construção civil apresentam, na maioria das vezes, a predominância de materiais inertes e passíveis de reaproveitamento, que não tem em grande parte uma finalidade. Esta dissertação apresenta uma abordagem sobre destinação correta dos resíduos, utilizando resíduos de argamassa, lodo de anodização de alumínio e cal para a formação de um novo compósito. O maior objetivo desta dissertação é caracterizar os materiais como componentes de novos compósitos para a construção civil, além de identificar novas composições (ao todo são 13 composições) atendendo as normas e leis existentes, analisar os processos físico-químicos, determinar as propriedades mecânicas e adaptar tecnologias existentes no processo de fabricação, além de destinar sem danos ambientais todas as matérias primas. Para análises físico-químicas e mecânicas foram estudados os seguintes parâmetros: resistência uniaxial média, absorção de água, densidade aparente, retração linear, microscopia eletrônica de varredura (MEV), espectrometria por energia dispersiva, difração de raios-X e espectrometria de fluorescência de raios-X (FRX). Dentre as 13 composições foram escolhidas as composições 4 e 11 para analisar mais o comportamento físico-químico. A resistência à compressão média uniaxial, aos 90 dias da composição 11 alcançou 9,18 MPa, com coeficiente de absorção de água de 12%, a retração foi de 1,53% e a densidade ao longo de 180 dias de cura variou apenas 4%. Os estudos físico-químicos identificaram a presença de Carbono, Sódio, Magnésio, Alumínio, Silício e Cálcio na composição 11, e através do MEV e FRX foi possível identificar que se trata de uma estrutura amorfa. Através desses estudos propõe-se alternativas que sejam revertidas em vantagens sociais e ambientais, tais como a fabricação de blocos vazados de concreto simples para alvenaria sem função estrutural, blocos cerâmicos para alvenaria de vedação, tijolos maciços para a alvenaria em relação a categoria A, entre outros. / Waste from the construction, have in most cases the predominance of inerts materials and insusceptible reuse, which has a few purpose. This dissertation presents an approach on the correct disposal of this waste, using mortar residue, anodizing aluminum sludge and lime to form a new composite. The main objective of this project is to characterize the materials as components of new composites for the construction industry, and identify new compositions (they are 13 compositions) meet the existing standards and laws, analyze the physical and chemical processes, determine the mechanical properties and adapting existing technologies in the manufacturing process, beyond to appropriate correctly without environmental damage for all the materials. For physical-chemical and mechanical analysis, the following parameters were studied: resistance average uniaxial, water absorption, apparent density, linear retraction, environment-scanning electron microscopy, energy dispersive x-ray, x-ray diffraction and x-ray fluorescence. Between the 13 compositions, chosen compositions 4 and 11 to analyze the behavior. The average uniaxial compressive strength at 90 days composition 11 reached 9,18 MPa with 12% water absorption coefficient, the shrinkage was 1,53% and the density over 180 days varied only 4%. The physic-chemical studies have identified the presence of Carbon, Sodium, Magnesium, Aluminum, Silicon and Calcium in the composition 11 and about scanning electron microscopy and XRF identified that it is an amorphous structure. Through these studies, it was possible to propose alternatives that are reversed in social and environmental benefits, such as the manufacture of simple concrete hollow blocks brickwork without structural function, ceramic blocks for sealing brickworks and solid bricks for brickworks in relation to category A, among others.

Pesquisa realizada para analisar o compósito de solo de decapagem (SD) considerado resíduos nas jazidas de minérios, em especial de calcário do município de Almirante Tamandaré, da Região Metropolitana de Curitiba neste trabalho, juntamente com o lodo e sedimentos de dragagem marinha do Porto de Antonina, no Estado do Paraná, e o uso cal virgem comum, como aglomerante pertencente à região de Almirante Tamandaré. Foram utilizados vários teores dos materiais para a determinação de 12 (doze) compósitos. Para o solo de decapagem os teores variaram entre 30% a 60%, para o material de dragagem os teores variaram entre 20% a 50% e o teor de aglomerante de cal virgem comum variou entre 15% a 30%. Foram moldadas idades de cura de 3 dias, 7, 14, 28, 60, 90, 180 e 365 dias. O aumento da resistência à compressão durante a cura foi proveniente das reações químicas das partículas sólidas das matérias-primas em ambiente alcalino gerando novas formações amorfas. Também foram analisados os materiais nos métodos de MEV com EDS, DRX e FRX, para a obtenção da composição química e formação estrutural dos novos compósitos. Todos os resultados da resistência à compressão uniaxial, com 28 dias de cura ao meio ambiente das 12 (doze) composições, obtiveram resultados médios de 7,7 MPa, atendendo satisfatoriamente a norma NBR 7170:1983 que determina a resistência à compressão igual ou acima de 4,0 MPa, destinada à produção de materiais de construção para alvenaria de vedação, sem fins estruturais. Comprovando o uso desses compósitos em materiais de construção civil, principalmente para os segmentos de blocos ou tijolos de vedações, sem fins estruturais, sem a necessidade de cozimento ou empreendimento de energia calorífica para a cura das peças, contribuindo para o meio ambiente e a sustentabilidade ambiental. / Research conducted to analyze the composite with pickling soil (SD), considered waste in mineral deposits, especially of limestone in the city of Almirante Tamandaré, the Metropolitan Region of Curitiba in this work, along with the sludge and marine dredging sediment from the Port of Antonina, State of Paraná, and use the common quicklime as binder belonging to the same region of Almirante Tamandaré. Various amounts of materials were used for the determination of twelve (12) composites. For the stripping soil content ranged from 30% and 60%, for the dredging material contents ranged from 20% and 50% and binder common quicklime content ranged from 15% to 30%. Were molded curing ages of 3 days, 7, 14, 28, 60, 90, 180 and 365 days. The increase in compressive strength during curing was derived from the chemical reactions of solid particles of raw materials in alkaline environment generating the new amorphous formations. Also the raw materials were analyzed in the SEM with EDS method, DRX and FRX, to obtain the chemical composition and formation structures of the new composite. All results of the uniaxial compressive strength, with 28 days of curing the environment of the twelve (12) compositions, obtained average results of 7.7 MPa, satisfactorily meeting the NBR 7170:1983 standard that determines the resistance to compression equal or above 4.0 MPa, for the production of building materials for sealing masonry without structural purposes. Proving the use of these composites in construction materials, especially for thread block or brick fences without structural purposes, without the need for cooking or heating energy project for the healing of parts, contributing to the environment and sustainability Environmental.

O crescimento populacional acelerado é o grande motivador para o desenvolvimento do setor da construção civil e o aumento na demanda por água potável, tendo como consequência, um aumento gradativo na geração de resíduos sólidos. Dessa forma, este trabalho foi realizado com o intuito de reciclar resíduos industriais e municipais incorporando-os em materiais para a construção civil. O compósito produzido a partir de lodo de estação de tratamento de água e lama de polimento de mármore, aplicando resíduo de produção de cal como ligante, foi avaliado quanto ao seu desempenho mecânico e sua estrutura morfológica. As matérias-primas foram caracterizadas quanto sua composição química, mineralógica,morfológica, granulométrica e, também, o teor de umidade. Com os materiais caracterizados, foram desenvolvidas nove composições variando-se o teor de lodo de estação de tratamento de água entre 25 e 50%, o teor de lama de polimento de mármore entre 35 e 50% e o resíduo de produção de cal entre 10 e 30%. Os compósitos foram submetidos a ensaios de resistência mecânica, absorção de água, análise química, mineralógica e morfológica. Os materiais desenvolvidos apresentaram, no 3° dia de cura, valor de resistência mecânica máxima de 4,65 MPa, no 7° dia 6,36 MPa, no 14° dia 6,74 MPa, no 28° dia 5,98 MPa, no 60° dia 8,52 MPa, no 90° dia 11,75 MPa e no 180° dia 12,06 MPa. Os valores de absorção de água aos 28 dias de cura variaram de 16,27 a 26,32% e aos 90 dias, de 13,57 a 23,56%. / The rapid population growth is the great motivator for the development of the construction industry and the increased demand for drinking water, resulting in a gradual increase in the generation of solid waste. Thus, this work was carried out in order to recycle industrial and municipal wastes incorporating them into materials for civil construction. The composite produced from water treatment sludge and marble polishing mud, applying lime production waste as a binder, was evaluated for its mechanical performance and its morphological structure. The raw materials were characterized for their chemical composition, mineralogy, morphology, particle size and also the moisture content. With the featured materials nine compositions have been developed varying the content of the water treatment sludge between 25 to 50%, marble polishing mud between 35 to 50% and the lime production waste between 10 to 30%. The composites were subjected to mechanical strength tests, water absorption, chemical and mineralogical composition and morphology. The developed materials presented, on the 3rd day of hydration, maximum strength value of 4.65 MPa, the 7th day 6.36 MPa, on the 14th day 6.74 MPa, the 28th day 5.98 MPa, on the 60th day 8.52 MPa at 90th day 11.75 MPa and 180th day 12.06 MPa. The water absorption values after 28 days of hydration ranged from 16.27% to 26.32% and after 90 days, from 13.57% to 23.56%.

The diploma thesis deals with the realization of a production and storage hall for the engineering industry. Part of the realization of the production hall is the demolition work of the existing agricultural building. The reconstruction of the existing premises of locksmith's workshop and dressing room for employees is also being implemented. For the preparation of this project, the thesis deals with the implementation of the main technological stages, technological plans for demolition work and substructure, transport routes of the main building materials and machines, technical report of the building site, time and financial plan of the construction, security measures especially for demolition works, quality control when drilling a pilot.

[ES] El descubrimiento del cemento Portland ha cambiado nuestra forma de construir, pero también es el responsable de grandes emisiones de CO2 a la atmósfera durante su fabricación (~1450 ᴼC), agravando la crisis actual que está sufriendo nuestro planeta debido al cambio climático y sus consecuencias en todo el medio ambiente. Por lo tanto, una alternativa más sostenible en la construcción es la utilización de la cal que necesita menor temperatura para su fabricación (~900 ᴼC). Si bien la introducción de puzolanas naturales o artificiales en morteros de cal han mejorado sus propiedades mecánicas y de durabilidad, estas aún tienen el inconveniente de ganar resistencias a edades largas de curado. Es por esta razón que en la presente tesis se pretende eliminar este inconveniente técnico, buscando la asociación de la cal con nuevos conglomerantes más sostenibles a partir de residuos para obtener morteros mixtos denominados cal/puzolana-geopolímero. Los residuos estudiados fueron: el catalizador gastado de craqueo catalítico, la ceniza de cascara de arroz, la tierra diatomea de origen residual y la ceniza de lodo de depuradora. También se estudia una puzolana natural proveniente de la República de Guatemala. En los morteros cal/puzolana (cal/FCC, cal/CCA, cal/CLD) se ha realizado sustituciones en peso hasta un 50 % de la mezcla cal/puzolana por geopolímero. El geopolímero se obtiene por una combinación del FCC como precursor y diferentes activadores alcalinos siendo estos la mezcla de: NaOH/Na2SiO3, NaOH/CCA, NaOH/TDN y NaOH/TDR. Los últimos tres sustituyen al silicato comercial como fuente de sílice alternativa. Asimismo, se realizaron estudios a nivel mecánico y microestructural. Para los estudios microestructurales, tanto de muestras endurecidas como de materiales de partida, se emplearon como técnicas: FRX, ADL, TG, DRX y FESEM. Los resultados han demostrado con éxito que añadir pequeñas cantidades de geopolímero sobre el sistema cal/puzolana fue notable, debido a que este potenció la formación de los nuevos productos de reacción, lo que mejoró la resistencia mecánica de los morteros desde las primeras horas de curado, llegando a obtener 7 veces más de resistencia que un mortero control cal/puzolana en 1 día de curado. El reemplazo del silicato de sodio comercial por CCA, TDN, TDR, como fuente de sílice, condujo a mejores desempeños del mortero en términos de resistencia a la compresión. Además, redujo el coeficiente de absorción de agua por capilaridad y aumento el tiempo de exposición a los ciclos hielo-deshielo frente a los morteros activados con activadores comerciales. Finalmente, los conglomerantes desarrollados en este estudio podría beneficiar tanto a la gestión de residuos como al desarrollo de materiales de construcción más sostenibles, aportando a los objetivos propuestos en la agenda 2030. / [CA] El descobriment del ciment Portland ha canviat la nostra manera de construir, però també és el responsable de grans emissions de CO¿ a l'atmosfera durant la seva fabricació (~1450 ᴼC), agreujant la crisi actual que està patint el nostre planeta degut al canvi climàtic i les seves conseqüències en tot el medi ambient. Per tant, una alternativa més sostenible en la construcció és la utilització de la calç que necessita menor temperatura per a la seva fabricació (~900 ᴼC). Si bé la introducció de putzolanes naturals o artificials en morters de calç han millorat les seves propietats mecàniques i de durabilitat, aquestes encara tenen l'inconvenient de guanyar resistències a edats llargues de curat. És per aquesta raó que en la present tesi es pretén eliminar aquest inconvenient tècnic, buscant l'associació de la calç amb nous conglomerants més sostenibles a partir de residus per a obtenir morters mixtos denominats calç/putzolana-geopolímer. Els residus estudiats van ser: el catalitzador gastat del craqueig catalític, la cendra de closca d'arròs, la terra diatomea d'origen residual i la cendra de llot de depuradora. També es va estudiar una putzolana natural provinent de la República de Guatemala. En els morters calç/putzolana (calç/FCC, calç/CCA, calç/CLD) s'ha realitzat substitucions en pes fins a un 50 % de la barreja calç/putzolana per geopolímer. El geopolímer s'obté per una combinació del FCC com a precursor i diferents activadors alcalins, sent aquests la mescla de: NaOH/Na2SiO3, NaOH/CCA, NaOH/TDN i NaOH/TDR. Els últims tres substitueixen al silicat comercial com a font de sílice alternativa. Així mateix, es van realitzar estudis a nivell mecànic i microestructural. Per als estudis microestructurals, tant de mostres endurides com de materia primera, es van emprar tècniques com: FRX, ADL, TG, DRX i FESEM. Els resultats han demostrat amb èxit que afegir petites quantitats de geopolímer sobre el sistema calç/putzolana va ser notable, pel fet que aquest va potenciar la formació dels nous productes de reacció, la qual cosa va millorar la resistència mecànica dels morters des de les primeres hores de curat, arribant a obtenir 7 vegades més de resistència que un morter control calç/putzolana en 1 dia de curat. El reemplaçament del silicat de sodi comercial per CCA, TDN, TDR, com a font de sílice, va conduir a millors acompliments del morter en termes de resistència a la compressió. A més, va reduir el coeficient d'absorció d'aigua per capil·laritat i va augmentar el temps d'exposició als cicles gel-desgel enfront dels morters activats amb activadors comercials. Finalment, els conglomerants desenvolupats en aquest estudi podrien beneficiar tant a la gestió de residus com al desenvolupament de materials de construcció més sostenibles, aportant als objectius proposats en l'Agenda 2030. / [EN] The discovery of Portland cement has changed the way we build; however, it is also responsible for large CO2 emissions into the atmosphere during its manufacturing (~ 1450 ᴼC), thereby aggravating the current crisis that our planet is suffering due to climate change and its consequences in the environment. Thus, a more sustainable alternative in construction is the use of lime that requires a lower temperature for its manufacturing (~ 900 ᴼC). The introduction of natural or artificial pozzolans in lime mortars has improved their mechanical properties and durability. Nevertheless, they present some technical disadvantages, as the low compressive strength, especially at early curing time. This thesis aims to eliminate this technical disadvantage by mixing lime with new more sustainable binders derived from waste, obtaining mixed mortars called lime/pozzolan-geopolymer The waste materials studied included fluid catalytic cracking residue (FCC), rice husk ash (CCA), residual diatomite (TDR) and sewage sludge ash (CLD). A natural pozzolan from the Republic of Guatemala was also studied. In the experimental procedure, in lime/pozzolan mortar mixtures (lime/FCC, lime/CCA, lime/CLD), up to 50% of their weight was substituted by geopolymer. The geopolymer is obtained by combining FCC as a precursor, and different alkaline activator mixtures including NaOH/Na2SiO3 (commercial waterglass), NaOH/CCA, NaOH/TDN, and NaOH/TDR, with the last three being an alternative silica source to commercial waterglass. In the same way, both mechanical and microstructural studies were carried out. The following techniques were used: XRF, ADL, TG, XRD and FESEM to assess the microstructural properties of both the raw materials and the hardened samples. The results have demonstrated that adding small amounts of geopolymer to the lime/pozzolan system was remarkable; it enhanced the formation of new reaction products, which improved the mechanical strength of the mortar from the first hours of curing, obtaining 7 times more strength than the lime/pozzolan control mortar in 1 day of curing. The replacement of commercial waterglass by CCA, TDN, TDR, as a source of silica, led to better performance of the mortar in terms of compressive strength. In addition, the coefficient of water absorption by capillarity was reduced and the exposure time during freezing-thawing cycles was increased compared to mortars activated with commercial activators. Finally, the binders developed in this study could benefit both waste management and the development of more sustainable construction materials, contributing to the objectives proposed in the 2030 Agenda. / Gracias al programa ADSIDEO-COOPERACIÓN de la Universitat Politècnica de València, que me ha brindado financiación para la presente investigación. / Villca Pozo, AR. (2021). Utilización de geopolímero para la mejora de las propiedades en morteros cal-puzolana y su empleo en países en desarrollo [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/172663 / TESIS

L'ingénierie enzymatique est un outil très avantageux dans l'industrie biotechnologique. Elle permet d'adapter les enzymes à une activité ou à une condition de réaction spécifique. En outre, elle peut permettre de déchiffrer les éléments clés qui ont facilité leur modification. Bien que l'ingénierie enzymatique soit largement pratiquée, elle comporte encore plusieurs goulets d'étranglement. Certains de ces goulets d'étranglement sont techniques, comme le développement de méthodologies pour la création de banques de mutations ciblées ou la réalisation de criblages à haut débit, et d'autres sont conceptuels, comme le déchiffrage des caractéristiques clés pertinentes d'une protéine cible pour la réussite d'un projet d'ingénierie. Parmi ces défis, l'épistasie intra-génique, ou la non-additivité des effets phénotypiques des mutations, est une caractéristique qui entrave grandement la prévisibilité. L'amélioration de l'ingénierie enzymatique nécessite une approche multidisciplinaire qui inclut une meilleure compréhension des relations structure-fonction-évolution. Cette thèse vise à contribuer à l'avancement de l'ingénierie enzymatique en étudiant deux systèmes modèles. Premièrement, des variantes dynamiques de la ß-lactamase TEM-1 ont été choisies pour étudier le lien entre la dynamique des protéines et l'évolution. La ß-lactamase TEM-1 a été largement caractérisée dans la littérature, ce qui s'est traduit par des connaissances approfondies sur son mécanisme de réaction, ses caractéristiques structurelles et son évolution. Les variantes de la ß-lactamase TEM-1 utilisées comme système modèle dans cette thèse ont été largement caractérisées, montrant une dynamique accrue à l'échelle temporelle pertinente pour la catalyse (µs à ms) mais maintenant la reconnaissance du substrat. Dans cette thèse, l'évolution in vitro de ces variantes dynamiques a été réalisée par des cycles itératifs de mutagenèse et de sélection aléatoires pour permettre une exploration impartiale du paysage de ‘fitness’. Nous démontrons que la présence de ces mouvements particuliers au début de l'évolution a permis d'accéder à des voies de mutations connues. De plus, des interactions épistatiques connues ont été introduites dans les variantes dynamiques. Leur caractérisation in silico et cinétique a révélé que les mouvements supplémentaires sur l'échelle de temps de la catalyse ont permis d'accéder à des conformations conduisant à une fonction améliorée, comme dans le TEM-1 natif. Dans l'ensemble, nous démontrons que l'évolution de la b-lactamase TEM-1 vers une nouvelle fonction est compatible avec divers mouvements à l'échelle de temps µs à ms. Il reste à savoir si cela peut se traduire par d'autres enzymes ayant un potentiel biotechnologique. Deuxièmement, la lipase Cal-A, pertinente sur le plan industriel, a été choisie pour identifier les caractéristiques qui pourraient faciliter son ingénierie. La lipase Cal-A présente des caractéristiques telles que la polyvalence du substrat et une grande stabilité thermique et réactivité qui la rendent attrayante pour la modification des triglycérides ou la synthèse de molécules pertinentes dans les industries alimentaire et pharmaceutique. Contrairement à TEM-1, la plupart des études d'évolution in vitro de la lipase Cal-A ont été réalisées dans un but industriel, avec une exploration limitée de l'espace de mutation. Par conséquent, les caractéristiques qui définissent la fonction de la lipase Cal-A restent insaisissables. Dans cette thèse, nous faisons état de la mutagenèse ciblée de la lipase Cal-A, confirmant l'existence d'une région clé pour la reconnaissance du substrat. Cela a été fait en combinant une nouvelle méthodologie de création de bibliothèque basée sur l'assemblage Golden-gate avec une visualisation structurelle basée sur des scripts pour identifier et cartographier les mutations sélectionnées dans la structure 3D. La caractérisation et la déconvolution de deux des plus aptes ont révélé l'existence d'une épistasie dans l'évolution de la lipase Cal-A vers une nouvelle fonction. Dans l'ensemble, nous démontrons que l’identification d'une variété de propriétés suite à la mutagenèse ciblée peut grandement améliorer la connaissance d'une enzyme. Cette information peut être appliquée pour améliorer l'efficacité de l'ingénierie dirigée. / Enzyme engineering is a tool with great utility in the biotechnological industry. It allows to tailor enzymes to a specific activity or reaction condition. In addition, it can allow to decipher key elements that facilitated their modification. While enzyme engineering is extensively practised, it still entails several bottlenecks. Some of these bottlenecks are technical such as the development of methodologies for creating targeted mutational libraries or performing high-throughput screening and some are conceptual such as deciphering the key relevant features in a target protein for a successful engineering project. Among these challenges, intragenic epistasis, or the non-additivity of the phenotypic effects of mutations, is a feature that greatly hinders predictability. Improving enzyme engineering needs a multidisciplinary approach that includes gaining a better understanding of structure-function-evolution relations. This thesis seeks to contribute in the advancement of enzyme engineering by investigating two model systems. First, dynamic variants of TEM-1 ß-lactamase were chosen to investigate the link between protein dynamics and evolution. TEM-1 ß-lactamase has been extensively characterized in the literature, which has translated into extensive knowledge on its reaction mechanism, structural features and evolution. The variants of TEM-1 ß-lactamase used as model system in this thesis had been extensively characterized, showing increased dynamics at the timescale relevant to catalysis (µs to ms) but maintaining substrate recognition. In this thesis, in vitro evolution of these dynamic variants was done by iterative rounds of random mutagenesis and selection to allow an unbiased exploration of the fitness landscape. We demonstrate that the presence of these particular motions at the outset of evolution allowed access to known mutational pathways. In addition, known epistatic interactions were introduced in the dynamic variants. Their in silico and kinetic characterization revealed that the additional motions on the timescale of catalysis allowed access to conformations leading to enhanced function, as in native TEM-1. Overall, we demonstrate that the evolution of TEM-1 b-lactamase toward new function is compatible with diverse motions at the µs to ms timescale. Whether this can be translated to other enzymes with biotechnological potential remains to be explored. Secondly, the industrially relevant Cal-A lipase was chosen to identify features that could facilitate its engineering. Cal-A lipase presents characteristics such as substrate versatility and high thermal stability and reactivity that make it attractive for modification of triglycerides or synthesis of relevant molecules in the food and pharmaceutical industries. Contrary to TEM-1, most in vitro evolution studies of Cal-A lipase have been done towards an industrially-specified goal, with limited exploration of mutational space. As a result, features that define function in Cal-A lipase remain elusive. In this thesis, we report on focused mutagenesis of Cal-A lipase, confirming the existence of a key region for substrate recognition. This was done by combining a novel library creation methodology based on Golden-gate assembly with script-based structural visualization to identify and map the selected mutations into the 3D structure. The characterization and deconvolution of two of the fittest revealed the existence of epistasis in the evolution of Cal-A lipase towards new function. Overall, we demonstrate that mapping a variety of properties following mutagenesis targeted to specific regions can greatly improve knowledge of an enzyme that can be applied to improve the efficiency of directed engineering.

Aiming for sustainable sanitation systems can provide benefits among a vast range of Sustainable Development Goals. In this study the sustainability of potential options for renovating or upgrading thewastewater treatment plant in Tupiza, a rapidly growing city in theSouthern highlands of Bolivia, was evaluated. The local context was characterized by increasing issues of flooding which in recent years has destroyed important wastewater treatment infrastructure and polluted sources of water for several downstream communities. Three system options consisting of different treatment technologies were evaluated against four criteria of sustainability; health,technical, environmental and financial and institutional. A "conventional" option consisting of waste stabilization ponds was compared against two more options with added steps of treatment, such as constructed wetlands, anaerobic reactors and alkaline and ammonia treatment of sludge. Social acceptance and demand of reuse of treated wastewater and sludge in agriculture was evaluated using qualitative research analysis. Results indicate that the systems with added treatment steps could help improve several areas of sustainability such as risks of disease transmission, space efficiency, treatment capacity and efficiency as well as enable safe reuse of sludge and wastewater in agriculture. Implementation of funding mechanisms covering the entire sanitation service chain as well as flood mitigation measures resulted essential in ensuring the long-term functionality of such improvements. This project was intended as a pre-study and identified several areas of future research including additional evaluation of nutrient content in effluent, investigation of a possible certification process for recycled byproducts from the wastewater treatment plant, risk assessment of floods of different magnitudes, evaluation of the long-term economic impact of having improved systems and evaluationof local institutional capacity surrounding the sanitation service chain in Tupiza. / Hållbara sanitetssystem för med sig många fördelar som kan främja majoriteten av de globala hållbarhetsmålen. I denna studie utreds hållbarheten i potentiella alternativ för renovering eller uppgradering av ett avloppsreningsverk i Tupiza, en snabbt växande stad i södra Bolivias högland. Den lokala kontexten präglades av en tilltagande översvämmningsproblematik som de senaste åren bidragit till förstörelse av central infrastruktur for avloppsvattenrening samt påföljande vattenföroreningar i samhällen nedströms reningsverket. Tre systemalternativ beståendes av olika reningstekniker utreddes utefter fyra hållbarhetskategorier; hälsa, teknologi, miljö samt finansiell/institutionell. Ett konventionellt alternativ som utgjordes av stabiliseringsdammar jämfördes mot två mer avancerade alternativ med ytterligare reningssteg såsom anlagd våtmark, anaerobiska reaktorer samt alkali- och ammoniakbehandling av slam. Social acceptans och efterfrågan för återanvändning av behandlat avloppsvatten och slam inom lantbruket utreddes i en kvalitativ forskningsstudie. Resultaten tyder på att system med fler reningssteg kan främja flera hållbarhetsområden såsom risk för överföring av sjukdomar, yteffektivitet, reningskapacitet och effektivitet samt möjliggöra säkert återanvändande av slam och avloppsvatten i jordbruket. Implementering av mekanismer för finansiering som täcker hela sanitetskedjan samt åtgärder mor översvämningar visade sig vara centrala för att säkerställa långsiktig funktion av sådana förbättringar. \\\\Det här projektet var menat som en förstudie och identifierade flertalet områden för vidare forskning såsom ytterligare utvärdering av näringsinnehåll i utgående vatten, utredning av möjlig certifieringsprocess för återvunna produkter från avloppsreningsverket, analys av översvämningsrisker av olika magnituder, utvärdering av den indirekta ekonomiska effekten av att ha system med fler reningssteg samt utvärdering av lokal institutionell kapacitet för hela servicekedjan för sanitet i Tupiza. / En este estudio se evaluó la sostenibilidad de opciones potenciales para renovar o mejorar la planta de tratamiento de aguas residuales de Tupiza, una ciudad de rápido crecimiento en las tierras altas del sur de Bolivia. En los últimos años, el contexto local se ha caracterizado por la ocurrencia de lluvias de mayor intensidad. Estas ocasionaron inundaciones que en efecto causaron daños importantes en la infraestructura de la planta de tratamiento de aguas residuales (PTAR). El deterioro de la infraestructura provocó el derrame de aguas residuales al río y la contaminación del agua en las comunidades ubicadas en la cuenca baja. Trés opciones de sistemas de tecnologías de tratamiento de aguas residuales para la renovación y/o mejoro de la planta de Tupiza fueron evaluadas considerando cuatro criterios de sostenibilidad: Salud, tecnolgía, ambiental y financiera/institucional. Por otra parte la aceptación social y demanda de reúso de aguas residuales y lodos tratados en la agricultura se evaluaron mediante un análisis de investigación cualitativa. La opción convencional que consiste en estanques de estabilización de desechos, se comparó con dos otras alternativas que incluyen pasos adicionales de tratamiento, como son humedales artificiales, reactores anaeróbicos y tratamiento de lodos con urea y cal. Los resultados indican que los sistemas de pasos adicionales de tratamiento podrían ayudar a mejorar diferentes aspectos de sostenibilidad, como los riesgos de transmisión de enfermedades, la eficiencia espacial,  la capacidad y eficiencia de tratamiento y la posibilidad de reutilización segura de lodos y aguas residuales en la agricultura. La implementación de mecanismos de financiación que cubren toda la cadena de servicios de saneamiento, así como las medidas de mitigacióin de inundaciones se mostraron esenciales para garantizar la funcionalidad a largo plazo de tales mejoras. La tesis es un estudio preliminar e identificó varias áreas de investigación futuras, la evaluación adicional del contenido de nutrientes en el efluente, la investigación de un posible proceso de certificación de subproductos reciclados de la PTAR, la evaluación de riesgos de inundaciones de diferentes magnitudes, la evaluación del impacto económico a largo plazo de la implementación de sistemas con pasos adicionales de tratamiento y la evaluación  de capacidad institucional local a lo largo de la cadena de servicios de sanemiento.