Les prophages sont des vecteurs majeurs de l’évolution des génomes bactériens et ont des rôles divers dans le processus adaptatif de leurs hôtes et peuvent leur apporter un avantage sélectif. Au cours de l’évolution, certains gènes prophagiques peuvent être perdus, notamment ceux codant pour des protéines du cycle lytique. Cependant, alors que certains de ces prophages défectifs sont capables de s’exciser, ils sont maintenus dans le génome de l’hôte, suggérant une pression sélective pour les conserver. C’est le cas du prophage défectif KplE1 chez E. coli K12. Dans l’équipe, des travaux ont mis en évidence que le maintien en lysogénie de différents prophages était sous le contrôle du terminateur de la transcription bactérien Rho. Afin d’identifier de nouveaux facteurs de l’hôte impliqués dans le maintien des prophages, j’ai développé un crible génétique qui m’a permis d’identifier plusieurs candidats impliqués dans le métabolisme général, la détoxification du NO ou qui appartiennent à un autre prophage défectif. Mon travail a été de discriminer lesquels de ces candidats jouaient un rôle significatif dans le maintien des prophages. Sur les trois gènes impliqués dans la détoxification du NO, seule l’expression de norV ou norW permet le maintien de KplE1. NorV réduit le NO et cette réduction nécessite l’utilisation d’un électron généré par l’oxydation du NADH par NorW. J’ai pu également montrer que l’expression du gène norV permettait le maintien d’un autre prophage fonctionnel (HK620) partageant le même module de recombinaison spécifique de site que KplE1. L’ensemble de mes résultats montre qu’il existe un lien co-évolutif important entre les prophages et leurs hôtes. / Prophages play recognized roles in their host genomes evolution and adaptation to variable ecosystems. They can provide to their host selective advantages that increase their competitiveness. Upon evolution, some prophage genes can be lost, especially those coding for lytic cycle capacity. While some of the defective prophages are perfectly competent for excision, they prove to be maintained in bacterial genomes, suggesting the involvement of a selective pressure. This is the case for our defective prophage model: KplE1 in E. coli K12. Previous work in our laboratory demonstrated that lysogeny maintenance of various prophages was controlled by Rho which is the bacterial transcription termination factor. In order to identify new host factors involved in prophage maintenance, I developed a genetic screen. This screen allowed me to identify candidate genes involved in bacterial general metabolism, in NO detoxification and also some genes that belong to another defective prophage. I determined which candidate genes actually played a role in KplE1 maintenance. Among the three genes involved in NO detoxification, I showed that norV or norW individual expression allowed KplE1 maintenance. NorV reduces NO and this reduction needs an electron produced by NorW NADH oxidation. I also showed that norV expression allowed the maintenance of another functional prophage (HK620) that shares the same site specific recombination module as KplE1. Together, my results illustrate the coevolution between prophages and their hosts.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This study used precision agriculture (AP) techniques to understand the dynamics of physical, chemical and biological soil for different winter management systems after three years of cultivation, to serve as the basis of the definition and indication of Intelligent Management Plans (PMI) in an efficient and practical way. The work was conducted in the city of Frederico Westphalen, RS, were evaluated ten winter management systems - fallow (PO), wild oat (Ap), white oat (AB), ryegrass (AZ), forage turnip (NF) vetch (E), white lupine (TB) and consortiums ApNFE, ApNF and ApE - with samples collected at the intersection points of a grid of 5 x 2.5 m, totaling 100 sampling points, so that there were ten points by sampling winter management system. The study was based on the evaluation of the effects of systems on the dynamics of spatial and horizontal distribution of physical, chemical and biological indicators of soil quality, an integrated analysis by proposing a soil quality index (IQS), providing support for future directions of PMI. In addition, it assessed the efficiency of the normalized difference vegetation index (NDVI) for estimating the production of the dry matter of winter management systems in order to provide a practical tool to assist in monitoring the operation of selected systems. The tools provided by AP facilitate the understanding of the dynamics of soil quality indicators in the tested systems, serving as a valuable tool in the definition and indication of PMI, providing greater knowledge of the ecology and behavior of soil biology, being possible to determine soil quality bioindicators. Based on the results, not recommended to opt for a system based in isolated benefits (indicators), requiring the joint evaluation of the variables, which was facilitated by the proposed IQS. In general, the systems that provide soil quality were ApNFE, ApE and NF, highlighting the facilitated root growth, in addition to greater nutrient supply afforded by these systems. The use of grass monocultures causes deterioration of chemical properties, due to low C/N and C/P ratios these systems, acting as nutrients immobilizers, must be rotated or consorted with other species over the years. The PO should be avoided in practice or even extinct, based on physical and biological degradation provided by this system. The NDVI was able to determine the dry matter produced by cover crops, except systems with visible flowers or consorted. The objectives proposed by this study were considered solved, since the answers obtained in the articles proposed by the latter, in order to make the multi-plant management or PMI proposition, more grounded and specialized. / Este trabalho usou de técnicas de agricultura de precisão (AP) para compreender a dinâmica de atributos físicos, químicos e biológicos do solo em função de diferentes sistemas de culturas de inverno após três anos de cultivo dos mesmos, visando servir como base na definição e indicação de Planos de Manejos Inteligentes (PMI) de forma eficiente e prática. O trabalho foi realizado no município de Frederico Westphalen, RS, sendo avaliados dez sistemas de culturas de inverno - pousio invernal (PO), aveia preta (Ap), aveia branca (AB), azevém (AZ), nabo forrageiro (NF), ervilhaca (E), tremoço branco (TB) e consórcios de ApNFE, ApNF e ApE -, com amostras coletadas nos pontos de cruzamento de uma malha de 5 x 2,5 m, totalizando 100 pontos de amostragem, de modo que houvessem dez pontos amostrais por sistema de cultura de inverno. O estudo baseou-se na avaliação dos efeitos de destes sistemas sobre a dinâmica de distribuição espacial e horizontal dos indicadores físicos, químicos e biológicos do solo, fazendo por fim, uma análise integrada através da proposição de um índice de qualidade do solo (IQS), dando suporte com embasamento firme para futuras indicações de PMI. Além disso, foi avaliada a eficiência do índice de vegetação por diferença normalizada (NDVI) na estimativa da produção de matéria seca pelos sistemas de culturas de inverno, de modo a fornecer uma ferramenta prática que auxilie no monitoramento do funcionamento dos sistemas escolhidos. Os instrumentos proporcionados pela AP facilitaram a compreensão da dinâmica dos indicadores da qualidade do solo nos sistemas testados, servindo como ferramenta valiosa na definição e indicação de PMI, além de ter proporcionado maior conhecimento da ecologia e comportamento da biologia do solo, sendo possível determinar bioindicadores da qualidade do solo. Com base nos resultados, não recomenda-se optar por determinado sistema baseado em benefícios isolados (indicadores), sendo necessária a avaliação conjunta das variáveis, o que foi facilitado pelo IQS proposto. De forma geral, os sistemas que proporcionaram maior qualidade do solo foram ApNFE, ApE e NF, destacando o crescimento radicular facilitado, além do maior suprimento de nutrientes propiciado por estes sistemas. O uso de gramíneas monocultivadas ocasiona a deterioração dos atributos químicos, devido a baixa relação C/N e C/P destas, funcionando como imobilizadores de nutrientes, devendo ser rotacionados ou consorciados com outras espécies ao longo dos anos. O PO deve ser evitado na prática ou até mesmo extinto, com base na degradação física e biológica proporcionada por este sistema. O NDVI foi capaz de determinar a matéria seca produzida pelos sistemas de forma satisfatória, com exceções de sistemas que apresentem flores visíveis, em consórcio. Os objetivos propostos ao início deste trabalho foram considerados sanados, visto as respostas obtidas nos artigos propostos por este, visando tornar a proposição de manejo multi-plantas ou PMI, mais fundamentada e especializada.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / With the advance the adoption of precision agriculture in Brazil, growing use the technological tools georeferenced soil sampling and crop yield. Relations and crossing data information between these two tools can assist in improving the management of crops through specific recommendations of each element with reduced costs, efficient use of inputs, increasing productivity and profitability of crops. The objective of this study was to evaluate the influence of soil chemical properties on grain yield of soybean and corn in two experimental fields southern Brazil through principal component analysis and hierarchical cluster. The research happened in two experimental areas of 132 and 124 hectares in typical Oxisol. The soil samples were taken with a grid sampling of one hectare in the 0-0.10 m layer. The yields of the cultures were related at the same point each soil analysis using the annual average productivity at a radius of 25 m around each sampling point. At the first experimental area of 132 hectares, were investigated yield maps soybean harvest crop 2008/09; soybean crop 2009/10; soybean crop 2010/11 and corn crop 2011/12. In the second study area of 124 hectares, were investigated yield maps soybean harvest crop 2009/10, corn harvest 2010/11 and soybean crop 2011/12. The selection of yield maps followed the criterion to evaluate crops with less than two years of the completion of soil sampling, where only occurred fertilization of crop maintenance. Both areas had high fertility, the first area to saturation (V) average 65.16% and 83.03% in the second area. At the first experimental area the increase in productivity is being explained by the increase of the attributes: saturation of calcium and magnesium in the cation exchange capacity (Sat Ca, Mg Sat), V, and the calcium: magnesium (Ca: Mg). In the second experimental area the increase attribute chemical Sat Mg committed crop yields. In both experiments the elevation of attributes Sat Ca, V and Ca: Mg contributed to raising crop yields. The balance of cations in the exchange complex was the chemical attributes that best explained the variability in crop investigated. / Com o avanço da adoção da agricultura de precisão no Brasil, cresce a utilização de ferramentas tecnológicas de amostragem de solo georreferenciadas e mapas de produtividade das culturas. As relações e o cruzamento de dados entre as informações destas duas ferramentas podem auxiliar no aprimoramento do manejo das lavouras através de indicações específicas de cada elemento com redução de custos, uso eficiente de insumos, aumento da produtividade e rentabilidade das culturas. O objetivo desse trabalho foi avaliar a influência de atributos químicos do solo na produtividade das culturas de soja e milho em duas áreas comerciais no planalto do RS, através da análise de componentes principais e o agrupamento hierárquico - cluster. A investigação ocorreu em duas áreas experimentais de 132 e 124 ha em Latossolo Vermelho distrófico típico. As amostragens de solo foram realizadas baseadas em uma malha regular de 100 metros por 100 metros na camada de 0-0,10 m. As produtividades das culturas foram relacionadas no mesmo ponto de cada análise de solo utilizando-se a média de produtividade anual em um raio de 25 m ao redor de cada ponto amostral. Na primeira área experimental de 132 ha, foram investigados os mapas de colheita de soja-safra 2008/09; soja-safra 2009/10; soja-safra 2010/11 e milho-safra 2011/12. Na segunda área investigada, de 124 ha, foram estudados os mapas de colheita de soja-safra 2009/10; milho-safra 2010/11 e soja-safra 2011/12. A escolha dos eventos de colheita analisados seguiu o critério de avaliar safras com período inferior a dois anos da realização da amostragem de solo, em que apenas ocorreram as adubações de manutenção das culturas. Ambas as áreas apresentavam fertilidade elevada, sendo a primeira com a saturação de bases (V) média de 65,16% e a segunda com 83,03%. Na primeira área experimental, o acréscimo de produtividade está sendo explicado pela elevação dos atributos: saturação de cálcio e de magnésio na capacidade de troca de cátions (Sat Ca; Sat Mg), V e relação cálcio: magnésio (Ca:Mg). Na segunda área experimental, a elevação do atributo químico Sat Mg comprometeu a produtividade das culturas. Em ambos os experimentos a elevação dos atributos Sat Ca, V e Ca: Mg contribuíram para elevar a produtividade das culturas. O equilíbrio de cátions no complexo de troca foi o atributo químico que melhor explicou a variabilidade da produtividade das culturas investigadas.

Plasma membrane transport proteins play a crucial role in all cells by conferring to the cell surface a selective permeability to a wide range of ions and small molecules. The activity of these transporters is often regulated by controlling their amount at the plasma membrane, via intracellular trafficking. The recent boom in the numbers of crystallized transporters shows that many of them that belong to different functional families with little sequence similarity adopt the same structural fold implying a conserved transport mechanism. These proteins belong to the APC (Amino acid-Polyamine-organoCation) superfamily and their fold is typified by the bacterial leucine transporter LeuT. This LeuT fold is characterized by inverted structural repeats of 5 transmembrane domains that harbor the central substrate-binding site and a pseudo-symmetry axis parallel to the membrane. The yeast Saccharomyces cerevisiae possesses about 16 amino acid permeases (yAAPs) that belong to the APC superfamily and that display various substrate specificity ranges and affinities. Topological, mutational analysis and in silico data indicate that yAAPS adopt the LeuT fold.<p><p>In this work we combined computational modeling and yeast genetics to study substrate binding by yAAPs and the endocytosis of these transporters in response to substrate transport. In the first part of this work, we analyzed the selective recognition of arginine by the yeast specific arginine permease, Can1. We constructed three-dimensional models of Can1 using as a template the recently resolved structure of AdiC, the bacterial arginine:agmatine antiporter, which is also a member of the APC superfamily. By comparison of the binding pockets of Can1 and Lyp1, the yeast specific lysine permease, we identified key residues that are involved in the recognition of the main and side chains of arginine. We first showed that the network of interactions of arginine in Can1 is similar to that of AdiC, and that the selective recognition of arginine is mediated by two residues: Asn 176 and Thr 456. Substituting these residues by their corresponding residues in Lyp1 converted Can1 into a specific lysine permease. In the second part of this work, we studied the regulation of two permeases, Can1 and the yeast general amino acid permease, Gap1. In the presence of their substrates, Gap1 and Can1 undergo ubiquitin-dependent endocytosis and targeting to the vacuolar lumen for degradation. We showed that this downregulation is not due to intracellular accumulation of the transported amino acids but to transport catalysis itself. By permease structural modeling, mutagenesis, and kinetic parameter analysis, we showed that Gap1 and Can1 need to switch to an intermediary conformational state and persist a minimal time in this state after binding the substrate to trigger their endocytosis. This down-regulation depends on the Rsp5 ubiquitin ligase and involves the recruitment of arrestin-like adaptors, resulting in the ubiquitylation and endocytosis of the permease.<p><p>Our work shows the importance of the structural analysis of yAAPs to get further insight into the different aspects of their function and regulation. We validate the use of a bacterial APC transporter, AdiC, to construct three-dimensional models of yAAPs that can be used to guide experimental analyses and to provide a molecular framework for data interpretation. Our results contribute to a better understating of the recognition mode of amino acids by their permeases, and the regulation of this transport in response to substrate binding. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Les vaccins conjugués furent développés suite à l’inefficacité des vaccins polysaccharidiques chez les nourrissons et les personnes âgées. Les vaccins conjugués sont composés d’un polysaccharide extrait de la capsule bactérienne et d’une protéine porteuse. Celle-ci permet de décupler la réponse immunitaire, permettant aux vaccins d’être efficaces. L’évolution des connaissances en chimie et en analytique permettent aujourd’hui de mieux caractériser ces vaccins et de mieux maîtriser leur production. Cependant, les chimies de conjugaison utilisées pour lier le polysaccharide et la protéine porteuse, ne sont pas toujours définies et cela mène souvent à l’obtention de produits hétérogènes. Les objectifs de cette thèse ont été d’étudier le polysaccharide, les protéines porteuses et de nouvelles voies de conjugaisons pour lier spécifiquement ces deux biomolécules.Différents outils analytiques ont été utilisés afin d’acquérir une meilleure connaissance des deux partenaires de conjugaison. Cela a également permis d’établir une stratégie d’analyse efficace pour caractériser les produits de réaction. La spécificité des réactions de conjugaison a été induite par l’utilisation d’espaceurs bi-fonctionnels, réagissant spécifiquement sur certains acides aminés. Leur réactivité a d’abord été testée sur un modèle peptidique. Cela a permis de faciliter la caractérisation et d’étudier l’efficacité et la spécificité des réactions. Les réactions efficaces ont ensuite été testées différents modèles : de la protéine au vaccin. Sur les quatre réactions testées, une a été efficace sur tous les modèles. Cette chimie de conjugaison est prometteuse pour le développement de nouveaux vaccins / Conjugate vaccines were developed because polysaccharide vaccines were not efficient in infant and old people. These vaccines were composed of the polysaccharide extracted from the bacterial capsule linked to a carrier protein. This protein created an immunological boost which allowed the vaccine to induce a proper protection for everyone. As chemistry knowledge and analytical techniques evolved, vaccines can now be better characterized and the production can be better controlled. Nevertheless, the chemistries used to bind the polysaccharide and the carrier protein are not always well-defined, which leads to the production of heterogeneous products. The objectives of this PhD were to study the polysaccharide, carrier proteins and new conjugation chemistries to specifically bind the two biomolecules. The other challenge was to be able to check the reaction specificity and characterize reaction products.To do so different analytical tools were used to allow a better knowledge of both conjugation partners but also to establish an efficient analytical strategy for glycoconjugate characterization. Conjugation reactions specificity was induced by using different bi-functional linkers, reacting specifically for one type of amino acid. Linkers’ reactivity was first tested on a model peptide. This allowed to facilitate the characterization and to check for both reaction specificity and reaction success. Efficient reactions were then tested on different models from carrier proteins to glycoconjugate vaccines. One of the four tested reactions was efficient from the peptide to the vaccine model. This conjugation is thus promising for the development of new conjugate vaccines

Dyer Island is thought to host one of the most abundant populations of white sharks on the planet; this is often credited to the large (55 – 60,000) Cape fur seal colony at Geyser Rock. Yet relatively little work has ever been produced from the area. This may be attributed to the harshness in its location as a study site, exposed to wind and swell from west to east which limits research periods. This study accounts for over 220 hrs of manual tracking at Dyer Island with a further 68 added from the inshore shallow areas of the bay. Sharks focused their movements and habitat use to reefs or channels that allowed access to Cape fur seals. Movement- Based Kernel Estimates (MKDE) were used to compute home range estimates for shark movements through and around the heterogeneous structures of Dyer Island and Geyser Rock. Inshore two core areas were revealed, one being the major reef system at Joubertsdam and the other at a kelp reef where the tracked shark had fed on a Cape fur seal. At Dyer Island one core area was identified in a narrow channel, ‘Shark Alley’, here a second tracked shark foraged for entire days within meters of rafting Cape fur seals. Rate of Movement (ROM) and Linearity (LI) of tracks were low during daytime and movements were focused around areas such as Shark Alley or other areas close to the seal colony before moving into deeper water or distant reefs with higher rates of ROM and LI at night. If moonlight was strong foraging would take place to the south of Geyser Rock but with higher ROM and LI than observed during the day. Foraging patterns in this study contrast studies from other sites in South Africa and home range and activity areas were comparatively much smaller than observed in Mossel Bay. It has been established that several known white sharks forage at Dyer Island and the other studied aggregation sites, such differences in foraging would suggest that they are able to adapt their foraging behaviour to suit the environment they are in; making them site specific in their foraging ecology. Both satellite and acoustic telemetry are revealing aggregation hotspots of white sharks in South Africa. It is important that such information is used to assist the recovery of the species which has been protected since 1991, yet is rarely considered in planning of coastal developments. / Dissertation (MSc)--University of Pretoria, 2012. / Zoology and Entomology / MSc / Unrestricted

[ES] La mejora genética vegetal tiene como objetivo la obtención de plantas con rasgos mejorados o características novedosas que podrían ayudar a superar los objetivos de sostenibilidad. Para este fin, la biotecnología vegetal necesita incorporar nuevas herramientas de ingeniería genética que combinen una mayor precisión con una mayor capacidad de mejora. Las herramientas de edición genética recientemente descubiertas basadas en la tecnología CRISPR/Cas9 han abierto el camino para modificar los genomas de las plantas con una precisión sin precedentes. Por otro lado, los nuevos enfoques de biología sintética basados en la modularidad y la estandarización de los elementos genéticos han permitido la construcción de dispositivos genéticos cada vez más complejos y refinados aplicados a la mejora genética vegetal. Con el objetivo final de expandir la caja de herramientas biotecnológicas para la mejora vegetal, esta tesis describe el desarrollo y la adaptación de dos nuevas herramientas: una nueva endonucleasa específica de sitio (SSN) y un interruptor genético modular para la regulación de la expresión transgénica. En una primera parte, esta tesis describe la adaptación de CRISPR/Cas12a para la expresión en plantas y compara la eficiencia de las variantes de Acidaminococcus (As) y Lachnospiraceae (Lb) Cas12a con Streptococcus pyogens Cas9 (SpCas9) descritos anteriormente en ocho loci de Nicotiana benthamiana usando expresión transitoria. LbCas12a mostró la actividad de mutagénesis promedio más alta en los loci analizados. Esta actividad también se confirmó en experimentos de transformación estable realizados en tres plantas modelo diferentes, a saber, N. benthamiana, Solanum lycopersicum y Arabidopsis thaliana. Para este último, los efectos mutagénicos colaterales fueron analizados en líneas segregantes sin la endonucleasa Cas12a, mediante secuenciación del genoma descartándose efectos indiscriminados. En conjunto, los resultados muestran que LbCas12a es una alternativa viable a SpCas9 para la edición genética en plantas. En una segunda parte, este trabajo describe un interruptor genético reversible destinado a controlar la expresión génica en plantas con mayor precisión que los sistemas inducibles tradicionales. Este interruptor, basado en el sistema de recombinación del fago PhiC31, fue construido como un dispositivo modular hecho de partes de ADN estándar y diseñado para controlar el estado transcripcional (encendido o apagado) de dos genes de interés mediante la inversión alternativa de un elemento regulador central de ADN. El estado del interruptor puede ser operado externa y reversiblemente por la acción de los actuadores de recombinación y su cinética, memoria y reversibilidad fueron ampliamente caracterizados en experimentos de transformación transitoria y estable en N. benthamiana. En conjunto, esta tesis muestra el diseño y la caracterización funcional de herramientas para la ingeniería del genómica y biología sintética de plantas que ahora ha sido completada con el sistema de edición genética CRISPR/Cas12a y un interruptor genético reversible y biestable basado en el sistema de recombinación del fago PhiC31. / [CAT] La millora genètica vegetal té com a objectiu l'obtenció de plantes amb trets millorats o característiques noves que podrien ajudar a superar els objectius de sostenibilitat. Amb aquesta finalitat, la biotecnologia vegetal necessita incorporar noves eines d'enginyeria genètica que combinen una major precisió amb una major capacitat de millora. Les eines d'edició genètica recentment descobertes basades en la tecnologia CRISPR/Cas9 han obert el camí per modificar els genomes de les plantes amb una precisió sense precedents. D'altra banda, els nous enfocaments de biologia sintètica basats en la modularitat i l'estandardització dels elements genètics han permès la construcció de dispositius genètics cada vegada més complexos i sofisticats aplicats a la millora genètica vegetal. Amb l'objectiu final d'expandir la caixa d'eines biotecnològiques per a la millora vegetal, aquesta tesi descriu el desenvolupament i l'adaptació de dues noves eines: una nova endonucleasa específica de lloc (SSN) i un interruptor genètic modular per a la regulació de l'expressió transgènica . En una primera part, aquesta tesi descriu l'adaptació de CRISPR/Cas12a per a l'expressió en plantes i compara l'eficiència de les variants de Acidaminococcus (As) i Lachnospiraceae (Lb) Cas12a amb la ben establida Streptococcus pyogens Cas9 (SpCas9), en vuit loci de Nicotiana benthamiana usant expressió transitòria. LbCas12a va mostrar l'activitat de mutagènesi mitjana més alta en els loci analitzats. Aquesta activitat també es va confirmar en experiments de transformació estable realitzats en tres plantes model diferents, a saber, N. benthamiana, Solanum lycopersicum i Arabidopsis thaliana. Per a aquest últim, els efectes mutagènics col·laterals van ser analitzats en línies segregants sense l'endonucleasa Cas12a, mitjançant seqüenciació completa del genoma i descartant efectes indiscriminats. En conjunt, els resultats mostren que LbCas12a és una alternativa viable a SpCas9 per a l'edició genètica en plantes. En una segona part, aquest treball descriu un interruptor genètic reversible destinat a controlar l'expressió gènica en plantes amb major precisió que els sistemes induïbles tradicionals. Aquest interruptor, basat en el sistema de recombinació del bacteriòfag PhiC31, va ser construït com un dispositiu modular fet de parts d'ADN estàndard i dissenyat per controlar l'estat transcripcional (encès o apagat) de dos gens d'interès mitjançant la inversió alternativa d'un element regulador central d'ADN. L'estat de l'interruptor pot ser operat externa i reversiblement per acció dels actuadors de recombinació i la seva cinètica, memòria i reversibilitat van ser àmpliament caracteritzats en experiments de transformació transitòria i estable en N. benthamiana. En conjunt, aquesta tesi mostra el disseny i la caracterització funcional d'eines per a l'enginyeria del genòmica i biologia sintètica de plantes que ara ha sigut completat amb el sistema d'edició genètica CRISPR/Cas12a i un interruptor genètic biestable i reversible basat en el sistema de recombinació del bacteriòfag PhiC31. / [EN] Plant breeding aims to provide plants with improved traits or novel features that could help to overcome sustainability goals. To this end, plant biotechnology needs to incorporate new genetic engineering tools that combine increased precision with higher breeding power. The recently discovered genome editing tools based on CRISPR/Cas9 technology have opened the way to modify plant¿s genomes with unprecedented precision. On the other hand, new synthetic biology approaches based on modularity and standardization of genetic elements have enabled the construction of increasingly complex and refined genetic devices applied to plant breeding. With the ultimate goal of expanding the toolbox of plant breeding techniques, this thesis describes the development and adaptation to plant systems of two new breeding tools: a site-specific nuclease (SSNs), and a modular gene switch for the regulation of transgene expression. In a first part, this thesis describes the adoption of the SSN CRISPR/Cas12a for plant expression and compares the efficiency of Acidaminococcus (As) and Lachnospiraceae (Lb) Cas12a variants with the previously described Streptococcus pyogens Cas9 (SpCas9) in eight Nicotiana benthamiana loci using transient expression experiments. LbCas12a showed highest average mutagenesis activity in the loci assayed. This activity was also confirmed in stable genome editing experiments performed in three different model plants, namely N. benthamiana, Solanum lycopersicum and Arabidopsis thaliana. For the latter, off-target effects in Cas12a-free segregating lines were discarded at genomic level by deep sequencing. Collectively, the results show that LbCas12a is a viable alternative to SpCas9 for plant genome engineering. In a second part, this work describes the engineering of a new reversible genetic switch aimed at controlling gene expression in plants with higher precision than traditional inducible systems. This switch, based on the bacteriophage PhiC31 recombination system, was built as a modular device made of standard DNA parts and designed to control the transcriptional state (on or off) of two genes of interest by alternative inversion of a central DNA regulatory element. The state of the switch can be externally and reversibly operated by the action of the recombination actuators and its kinetics, memory, and reversibility were extensively characterized in N. benthamiana using both transient expression and stable transgenics. Altogether, this thesis shows the design and functional characterization of refined tools for genome engineering and synthetic biology in plants that now has been expanded with the CRISPR/Cas12a gene editing system and the phage PhiC31-based toggle switch. / Bernabé Orts, JM. (2019). Development and characterization of two new tools for plant genetic engineering: A CRISPR/Cas12a-based mutagenesis system and a PhiC31-based gene switch [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/133055 / TESIS

Vondra, M. (2011). The influence of the environment on the art work, environmental art as an inspiration for the ecological and art projects with children, diploma work. Prague: Charles University, Faculty of education, art department, 104 pages. (Attachments out of the book binding - set of 12 paintings, 4 graphic lists and set of 16 photos) This thesis is dealing with the matter of infuence of environment on the art work. It is analyzing differencies in the understanding of the environmental art term, and its historical bases. The thesis is comparing two different cultures - finish and czech, concerning its way of using the term. The comparison show the problematic issue of the czech prehension of the environmental art term. Didactic section presents a project of the mountain leasure time centre focused on the ecological education through the art activities in the nature.

L'état circulaire du chromosome bactérien pose un problème particulier lors de la réplication. Un nombre impair d'événements de recombinaison homologue donne des chromosomes dimères concaténés qui ne peuvent pas être divisés en cellules filles. Pour résoudre ce problème, les bactéries ont mis au point un mécanisme de résolution des dimères basé sur un système de recombinaison spécifique au site. Ceci est effectué par le système Xer/dif. Dans ce système, les protéines Xer effectuent une réaction de recombinaison dans le site dif au niveau du septum cellulaire immédiatement avant la division cellulaire. Dans la plupart des bactéries, cette réaction est effectuée par deux recombinases, XerC et XerD. Cependant, Streptococcus suis, un agent pathogène zoonotique important utilise un système de recombinaison différent, constitué d'une seule enzyme recombinase appelée XerS, qui catalyse la réaction de recombinaison dans un site dif non conventionnel. Pour caractériser le mode de clivage de XerS, des expériences EMSA ont été réalisées en utilisant des fragments de PCR marqués par HEX et des "suicide substrates". Nos données suggèrent que 1.) XerS est capable de lier la séquence entière de difSL; 2.) XerS lie plus efficacement le côté gauche des mutants difSL incomplets que le côté droit; 3.) XerS coupe les brins supérieur et inférieur du site difSL, avec une réaction plus efficace au bas. 4.) Modifications des nucléotides de la région la plus externe ou de la région centrale changent les préférences de clivage. 5.) XerS n'a montré aucune activité spécifique sur un autre site dif non conventionnel des Firmicutes, 6.) XerS interagit avec la sous-unité FtsK-y. L'ensemble des résultats présentés permet de mieux comprendre le fonctionnement de la recombinaison XerS dans le système de recombinase unique de Streptococcus et comment cette recombinaison est régulée par des facteurs de l'hôte. / The circular state of the bacterial chromosome presents a specific problem during replication. An odd number of homologous recombination events results in concatenated dimer chromosomes that cannot be partitioned into daughter cells. To solve this problem, bacteria have developed a mechanism of dimer resolution based on site-specific recombination system. This is performed by the Xer/dif system. In this system, the Xer proteins perform a recombination reaction in the dif site at the cell septum immediately prior to cell division. In most bacteria this reaction is performed by two recombinases, XerC and XerD. However, an important zoonotic pathogen; Streptococcus suis harbors a different recombination system, composed by a single recombinase enzyme called XerS, that catalyzes the recombination reaction in an unconventional dif site; difSL. A region characterized by two imperfect inverted repeat regions that flank a central region of 11 bp.To characterize the mode of cleavage of XerS, EMSA experiments were performed by using HEX-labelled PCR fragments and “nicked suicide substrates”. Our data suggests that; 1.) XerS is able to bind the entire difSL sequence; 2.) XerS binds more efficiently the left half side on incomplete difSL mutants than the right half side; 3.) XerS cleaves both the top and bottom strands of the difSL site, with a more efficient reaction at the bottom strand; 4.) Nucleotides at the outermost region of a T rich region seem to be determinant for binding selectivity and modifications of the extra spacing between the inverted repeat arms as well as length modifications of the central region change cleavage preference. 5.) XerS did not show any specific activity on another unconventional dif site in Firmicutes, as tested on difH. 6.) XerS interacts with FtsK-y subunit. This research aims to understand how XerS recombination works in the single recombinase system of Streptococcus and how this recombination is regulated by host factors. Exploration of these recombinases will provide a better understanding of the mechanisms of DNA exchange and genome stability in bacteria. It can also increase our knowledge of the evolution and speciation of recombinogenic bacteria.

Ein kontinuierlich ansteigender Wasserbedarf, verursacht durch verstärktes Bevölkerungswachstum, zunehmende Urbanisierung und Industrialisierung, einhergehend mit einer Übernutzung der verfügbaren Wasserressourcen, führt weltweit zu einem dauerhaften Absinken der Grundwasserstände. Um das zeitliche Ungleichgewicht zwischen lokalem Wasserbedarf und Verfügbarkeit zu überwinden und die daraus resultierenden negativen Auswirkungen abzumildern, erfolgt im Rahmen einer künstlichen Grundwasseranreicherung die gezielte Anreicherung oder Wiederaufladung eines Aquifers. Dazu wird überschüssiges Oberflächenwasser unter kontrollierten Bedingungen versickert oder infiltriert, um es in Zeiten von Wassermangel zur Verfügung zu stellen oder die ökologischen Randbedingungen zu verbessern. Beim Betrieb der dafür häufig eingesetzten Infiltrationsbecken kommt es in Abhängigkeit von den Standort- (Boden/Klima/Wasserqualität) und den Betriebsparametern (Hydraulische Beladungsrate, Hydraulischer Beladungszyklus) allerdings durch verschiedene Prozesse (Kolmation, Sauerstoff- und Nährstofftransport) häufig zur negativen Beeinflussung der quantitativen und qualitativen Effizienz solcher Anlagen. Bisher durchgeführte Untersuchungen im Labor- und Feldmaßstab sowie die im Zuge des Betriebes bestehender Infiltrationsbecken gewonnenen Daten liefern hauptsächlich Informationen zum Einfluss einzelner Randbedingungen auf die Veränderung der Infiltrationskapazität bzw. die quantitative Effizienz. Allerdings können auf Basis dieser Daten nicht alle offenen Fragen hinsichtlich des Einflusses der Standort- und Betriebsparameter auf die quantitative und qualitative Effizienz von Infiltrationsbecken vollumfänglich und abschließend beantwortet werden. Aufgrund nicht untersuchter Aspekte sowie widersprüchlicher Daten existieren Unsicherheiten bezüglich der Bewertung hinsichtlich des Einflusses der einzelnen Standort- und Betriebsparameter auf die Effizienz solcher Anlagen. Zur Generierung von weiterem Wissen über den Einfluss von Standort- und Betriebsparametern auf die Effizienz von Infiltrationsbecken und zur anschließenden Formulierung von Empfehlungen für eine optimierte Standortauswahl sowie Betriebsweise von Infiltrationsbecken erfolgt die Durchführung von Laborversuchen mittels kleinskaliger und großskaliger, physikalischer Modelle. Es werden verschiedene Infiltrationsszenarien bei wechselnden Randbedingungen (Bodenart, Temperatur, Wasserqualität, Hydraulische Beladungsrate, Hydraulischer Beladungszyklus) durchgeführt. Anhand der gewonnenen Daten kann die Beeinflussung der quantitativen und qualitativen Effizienz durch die verschiedenen Standort- und Betriebsparameter sowie die dadurch beeinflussten Prozesse sehr gut aufgezeigt werden. Das bisher existierende Wissen kann dabei zum Teil bestätigt und um zusätzliche Erkenntnisse erweitert werden. Es zeigt sich, dass eine höhere hydraulische Durchlässigkeit des anstehenden Bodens eine geringere Reduzierung der Infiltrationskapazität durch Kolmationsprozesse verursacht und zudem für eine bessere Sauerstoffverfügbarkeit sorgt. Darüber hinaus wird ersichtlich, dass Bodentexturen mit einem mittleren Porendurchmesser von 230 µm optimale Bedingungen für eine hohe biologische Aktivität einhergehend mit einem Abbau infiltrierter Substanzen bieten. Der Nachweis einer verstärkten Reduzierung der Infiltrationskapazität durch Kolmationsprozesse bei erhöhten Temperaturen, aber nicht vorhandener Sonneneinstrahlung, kann nicht erbracht werden, da das Fließen des infiltrierten Wassers signifikant durch die erhöhte Viskosität beeinflusst wird. Eine schlechtere Wasserqualität, gleichbedeutend mit erhöhten Konzentrationen an abfiltrierbaren Stoffen sowie gelöstem organischen Kohlenstoff, verursacht in den simulierten Infiltrationsszenarien eine stärkere Reduzierung der Infiltrationskapazität. Die physikalischen Kolmationsprozesse tragen dabei den Hauptanteil an der Reduzierung der Infiltrationskapazität. Des Weiteren wird nachgewiesen, dass eine erhöhte HBR zu einer verstärkten Reduzierung der Infiltrationskapazität und zu einer verschlechterten Sauerstoffverfügbarkeit führt. Die Länge der Infiltrations- und Trockenphasen während des simulierten Betriebes von Infiltrationsbecken beeinflusst entscheidend die Reduzierung der Infiltrationskapazität sowie die Sauerstoffverfügbarkeit. Dabei kann gezeigt werden, dass unabhängig von der Länge der Infiltrations- und Trockenphasen eine vollständige Wiederherstellung der Sauerstoffverfügbarkeit innerhalb von 24 h im Anschluss an eine Infiltrationsphase gewährleistet wird. Das Verhältnis von Infiltrations- und Trockenphasen, auch als Hydraulischer Beladungszyklus bezeichnet, hat hingegen nahezu keinen Einfluss auf die quantitative Effizienz. Bei der Betrachtung aller simulierten Infiltrationsszenarien inklusive der Wechselwirkungen zwischen den verschiedenen Standort- und Betriebsparametern können die optimalen Bedingungen für eine hohe quantitative und qualitative Effizienz von Infiltrationsbecken identifiziert werden. Diese sind gegeben beim Vorhandensein eines gut durchlässigen Bodens (hydraulische Leitfähigkeit > 10-4 m s-1), idealerweise mit einem mittleren Porendurchmesser von 230 µm, gepaart mit einer intermittierenden Infiltration von Wasser höherer Qualität ((AFS ≤ 10 mg L-1, BDOC ≤ 10 mg L-1) und der Vermeidung von Infiltrationsphasen länger als 24 h. Eine Widerspiegelung der experimentellen Ergebnisse sowie eine Vorhersage der Reduzierung der Infiltrationskapazität ist mit dem ausgewählten, analytischen Modell nach Pedretti et al., 2012 aufgrund der unzureichend implementierten Berücksichtigung veränderlicher Eingangsparameter nur bedingt möglich. Auf Basis der gewonnenen Daten und dem damit einhergehenden erweiterten Wissen über den Einfluss von Standort- und Betriebsparametern auf die Effizienz von Infiltrationsbecken können schlussendlich Empfehlungen für die Standortauswahl und die optimale Betriebsweise ausgesprochen werden.:1 Einleitung...1 2 Grundlagen der künstlichen Grundwasseranreicherung...7 3 Vorliegende Erkenntnisse zur Beeinflussung der quantitativen und qualitativen Effizienz durch Standort- und Betriebsparameter...38 4 Methoden...49 5 Gewonnene Erkenntnisse hinsichtlich der Beeinflussung der quantitativen und qualitativen Effizienz durch Standort- und Betriebsparameter...87 6 Empfehlungen zur Optimierung von Standort- und Betriebsbedingungen von Infiltrationsbecken zur künstlichen Grundwasseranreicherung...128 7 Schlussfolgerung und Ausblick...136 / A continuously rising demand for water, caused by increased population growth, growing urbanization and industrialization, accompanied by overuse of available water resources, is leading to a permanent drop in groundwater levels worldwide. In order to overcome the temporal imbalance between local water demand and availability and to mitigate the resulting negative effects, artificial groundwater recharge involves the managed enrichment or recharging of an aquifer. For this purpose, excess surface water is percolated or infiltrated under controlled conditions in order to make it available in times of water shortage or to improve the ecological boundary conditions. However, the quantitative and qualitative efficiency of frequently used infiltration basins during the operation is often negatively influenced by a wide variety of processes (clogging, oxygen and nutrient transport), depending on the location (soil/climate/water quality) and the operating parameters (loading rate, loading cycle). Investigations conducted to date on laboratory and field scale as well as data obtained during the operation of existing infiltration basins provide information on the influence of individual boundary conditions on the change in infiltration capacity or quantitative efficiency. However, not all open questions regarding the influence of site specific and operating parameters on the quantitative and qualitative efficiency of infiltration tanks can be answered completely and conclusively on the basis of these data. Due to aspects that have not been investigated and contradictory data, there are uncertainties in the evaluation regarding the influence of the individual site and operating parameters on the efficiency of the plants. Laboratory tests using small-scale and large-scale physical models were carried out, in order to generate further knowledge about the influence of site specific and operating parameters on the efficiency of infiltration basins and to formulate subsequently recommendations for an optimised site selection and operation of these plants. Various infiltration scenarios were carried out under changing boundary conditions (soil type, temperature, water quality, hydraulic loading rate, hydraulic loading cycle). Based on the data obtained, the influence on the quantitative and qualitative efficiency by the various site specific and operating parameters and the processes influenced by them can be demonstrated very well. The existing knowledge can be partially confirmed and extended by additional findings. It shows that a higher hydraulic permeability of the existing soil causes a lower reduction of the infiltration capacity by clogging processes and provides also a better oxygen availability. Furthermore, it can be observed that soil textures with an average pore diameter of 230 µm offer optimal conditions for high biological activity combined with a strong degradation of infiltrated substances. In case of higher temperatures but without solar radiation, an increased reduction of the infiltration capacity by clogging processes cannot be observed, since the flow of the infiltrated water is significantly influenced by the increased viscosity. In the simulated infiltration scenarios, poorer water quality, synonymous with increased concentrations of filterable substances as well as dissolved organic carbon, cause a stronger reduction of the infiltration capacity. Physical clogging processes are contributing the major part to the reduction of the infiltration capacity. Furthermore, it can be shown that an increased hydraulic loading rate leads to an increased reduction of the infiltration capacity and to a decreased oxygen availability. The length of the infiltration and drying phases during the simulated operation of infiltration basins has a decisive influence on the reduction of the infiltration capacity and the oxygen availability. It is demonstrated that regardless of the length of the infiltration and drying phases, a complete restoration of oxygen availability can be guaranteed within 24 h following an infiltration phase. In contrast, the ratio of infiltration and dry phases, also known as the hydraulic loading cycle, has almost no influence on the quantitative efficiency. Optimal conditions for a high quantitative and qualitative efficiency of infiltration basins can be identified, when considering all simulated infiltration scenarios including the interactions between the different site specific and operating parameters. These are given in the presence of a well-permeable soil (hydraulic conductivity > 10-4 m s-1), ideally with an average pore diameter of 230 µm, coupled with an intermittent infiltration of water of higher quality ((AFS ≤ 10 mg L-1, BDOC ≤ 10 mg L-1) and the prevention of infiltration phases longer than 24 h. A reflection of the experimental results as well as a prediction of the reduction of the infiltration capacity with the selected analytical model according to Pedretti et al., 2012 is only conditionally possible due to the insufficiently implemented consideration of variable input parameters. Recommendations for site selection and optimal operation were finally made on the basis of the data obtained and the resulting extended knowledge about the influence of site specific and operating parameters on the efficiency of infiltration basins.:1 Einleitung...1 2 Grundlagen der künstlichen Grundwasseranreicherung...7 3 Vorliegende Erkenntnisse zur Beeinflussung der quantitativen und qualitativen Effizienz durch Standort- und Betriebsparameter...38 4 Methoden...49 5 Gewonnene Erkenntnisse hinsichtlich der Beeinflussung der quantitativen und qualitativen Effizienz durch Standort- und Betriebsparameter...87 6 Empfehlungen zur Optimierung von Standort- und Betriebsbedingungen von Infiltrationsbecken zur künstlichen Grundwasseranreicherung...128 7 Schlussfolgerung und Ausblick...136