Le rhabdomyosarcome (RMS) est le cancer des tissus mous le plus courant chez l'enfant, et moins de 30 % des patients à haut risque obtiennent une rémission. Par conséquent, il existe un besoin pour une immunothérapie nouvelle et efficace. Les cellules tueuses naturelles (NK), avec leur capacité intrinsèque à tuer les cellules cancéreuses, représentent un outil thérapeutique prometteur. Cependant, leur efficacité clinique est limitée. Ainsi, nous proposons de concevoir ces cellules avec un récepteur antigénique chimérique (CAR) qui permettra aux cellules NK de cibler plus efficacement les cellules de RMS. De plus, nous proposons aussi de concevoir grâce à la technologie CRISPR-Cas9, des NK n’exprimant pas NKG2A, un récepteur impliqué dans l'inhibition des cellules NK par le microenvironnement tumoral. Nous avons développé un vecteur lentiviral codant pour une construction CAR reconnaissant B7-H3 et FGFR4, deux protéines surexprimées à la surface des cellules RMS, associées à une queue intracellulaire optimisée pour l'activation des NK. Les cellules NK primaires expandues ont été transduites et triées en fonction de l'expression du CAR, conduisant à une population de cellules CAR+- NK enrichie. L'efficacité des deux CAR a été évaluée par des tests cytotoxiques et de dégranulation contre les lignées cellulaires de RMS, RH-30 et RD, toutes deux exprimant B7-H3 et FGFR4. Les résultats préliminaires ont montré une augmentation de la cytotoxicité de 20 % par rapport aux NK de type sauvage pour les CAR anti-B7-H3. Les cellules NK ont également été transduites pour éliminer l’expression du gène KLRC1, codant pour NKG2A, en utilisant CRISPR Cas9. Ceci a permis d’augmenter la cytotoxicité des NK de 20% à 25% comparativement aux NK qui expriment NKG2A. Nous avons aussi combiné les deux modifications génétiques, obtenant ainsi des NK qui expriment un CAR contre les cellules du RMS et n’exprimant pas NKG2A. Des résultats préliminaires nous ont permis d’observer que les NK doublement modifiées étaient 60% plus cytotoxiques que les NK non-transduites et 20% plus efficaces que les CAR-NK ou les NK n’exprimant pas NKG2A. Ce projet sera une preuve de principe qu'une thérapie hautement innovante basée sur l'ingénierie des cellules NK est efficace et applicable au cancer solide. / Rhabdomyosarcoma (RMS) is the most common soft tissue cancer in childhood, and less than 30% of high-risk patients achieve remission. Therefore, there is a need for new and efficient immunotherapy. Natural killer (NK) cells, with their intrinsic ability to kill cancer cells, represent a promising therapeutic tool. However, their clinical efficacy is limited. Thus, we propose to engineer these cells with a Chimeric Antigen Receptor (CAR) that will allow NK cells to target RMS cells more efficiently and though the knock-out of NKG2A, a receptor involved in the inhibition of NK cells by the tumor microenvironment. We developed a lentiviral vector coding for a CAR construct recognizing B7-H3 and FGFR4, two proteins overexpressed at the surface of RMS cells, combined to an intracellular tail optimized for NK activation. Expanded primary NK cells were transduced and sorted based on CAR expression, leading to an enriched CAR+ -NK cells population. Efficacy of both CARs was evaluated by cytotoxic and degranulation assays against RH-30 and RD RMS cell lines, both expressing B7-H3 and FGFR4. Preliminary results showed an increase in cytotoxicity of 20% compared to wild type NK for CAR anti-B7-H3. NK cells were also knocked-out for the gene coding for NKG2A, using CRISPR Cas9, thereby increasing cytotoxicity by 20% to 25%. The combination of both genetic modifications should significantly increase the efficacy of NK-cell based therapy in RMS. Indeed, preliminary results allowed us to observe that doubly modified NKs were more than 60% more cytotoxic than non-transduced NKs and 20% more effective than CAR-NKs or NKs not expressing NKG2A. This project will be a proof of principle that a highly innovative therapy based on NK-cell engineering is efficient and applicable to solid cancer.

The waters of the open ocean constitute the largest living space on Earth but despite its obvious significance to the biosphere, the open ocean remains an unexplored frontier. With a regional focus on the Indian Ocean, this thesis investigates (i) the distribution of pelagic biota on basin scales, (ii) the effect of abrupt topography on pelagic biota and their predator-prey relationships, and (iii) the use of genetic techniques to elucidate population connectivity and dispersal of pelagic taxa. (i) Pelagic scattering layers (SLs) were surveyed with scientific echosounders across the southwest (SWIO) and central Indian Ocean to investigate their vertical and geographical distribution. Structurally distinct SL regimes were found across the Subantarctic Front, and may explain recently observed foraging behaviours of southern elephant seals. Regression models indicated a close relationship between sea surface temperature and mean volume backscatter, with significantly elevated backscatter in the subtropical convergence zone. The heterogeneous distribution of scattering layer biota may have implications for predator foraging and carbon cycling in the Indian Ocean. (ii) Acoustic surveys revealed diverse interactions between SLs, aggregations and topography around islands as well as shallow ( < 200m) and intermediate (200-800m) seamounts at spatial scales from 1 to 100 km. Epi-and mesopelagic backscatter was increased around reefs and banks of the Chagos archipelago, indicating connectivity between oceanic and neritic systems. SWIO seamounts harboured summit-associated aggregations, but the distributions of surrounding SLs did not follow a general pattern. Downstream SL depletion was observed in one location and combined with stomach content analyses, provides an insight into the mechanics of prey flux between open-ocean and seamount ecosystems. (iii) A mitochondrial marker was used to assess the population structure and demography of the hatchetfish Argyropelecus aculeatus in the SWIO. The results are suggestive of a single, well-connected population and indicate a recent population expansion around 0.14 million years ago. This highlights that even highly abundant mesopelagic populations are vulnerable to global climatic changes. Dispersal and recruitment are key ecological processes structuring seamount communities and are directly relevant for the management of exploited populations. Genetic barcoding was evaluated as a means to identify cryptic larval specimens of eels (leptocephali) and spiny lobsters (phyllosomata). Identification success was limited, but indicated the presence of 3-4 phyllosoma clades and 5-6 leptocephalus clades along the SWIR.

This thesis examines the notions of time and epoch through the works of three contemporary French authors: Christine Montalbetti, Hélène Lenoir and Emmanuelle Pireyre. The theoretical framework for this study draws upon literary criticism, time studies and cultural theory: it investigates in particular the ways in which literary fiction may respond to what has been called a ‘culture of speed' in capitalist economies of the twenty-first century. This culture of speed is traced back two major epochal shifts: the revolution in information technology, which has permitted the generating and sharing of information at exponentially higher speeds, and an increasing consciousness of the vast time cycles within which we might situate our own epoch or individual lives. This work considers the ways in which this collective and paradigmatic shift might be reflected in literary fiction. It examines the representation of new information technologies within these literary works, focusing in particular on the texts' representations of obsessive or compulsive uses of technology and the kinds of anxieties emerging as a result of the ubiquity of these devices. It further questions whether new aesthetic trends, what has been called a ‘post-internet aesthetic', may be emerging in literary fiction in light of some of these changes. Further investigation of the representation of diegetic time within these texts demonstrates that these literary works appear to resist the current time culture of speed and simultaneity, embracing instead the literary devices of repetition and digression while maintaining a dilatory pace. This study also considers the emergence of ‘short-termism' and insularity within these literary texts as reflecting a wider societal trend, especially in light of recent theoretical work on the vast timescales (for example those of the planet's climate cycles) that have become increasingly present in political and journalistic discourses.

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-08-30T11:53:59Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_ReconstrucaoModelagemSilico.pdf: 1615944 bytes, checksum: fd70334c4cf9d3608beb1b9294e63cdf (MD5) / Approved for entry into archive by Irvana Coutinho (irvana@ufpa.br) on 2017-08-30T16:33:29Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_ReconstrucaoModelagemSilico.pdf: 1615944 bytes, checksum: fd70334c4cf9d3608beb1b9294e63cdf (MD5) / Made available in DSpace on 2017-08-30T16:33:29Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_ReconstrucaoModelagemSilico.pdf: 1615944 bytes, checksum: fd70334c4cf9d3608beb1b9294e63cdf (MD5) Previous issue date: 2016-04-04 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / A modelagem matemática in silico baseada em restrições é uma abordagem adotada pela Biologia de Sistemas para analisar redes metabólicas. A bactéria Gram-positiva Exiguobacterium antarticum B7 é um extremófilo capaz de sobreviver em ambientes frios como gelo glacial e permafrost. A capacidade de adaptação ao frio desses micro-organismos vem despertando grande interesse biotecnológico. Um fator importante para o entendimento do processo de adaptação ao frio está relacionado à modificação química de ácidos graxos que constituem a membrana celular das bactérias psicotróficas. A finalidade é manter a fluidez da membrana para evitar o congelamento da bactéria. Neste trabalho, a via metabólica de biossíntese de ácidos graxos da bactéria E. antarticum B7 foi reconstruída a partir do genoma anotado disponível. As ferramentas de software KEGG (Kyoto Encyclopedia of Genes and Genomes) e RAST (The Rapid Annotation Server) foram utilizadas para gerar o modelo preliminar da rede. O passo seguinte foi a etapa de cura manual baseada na combinação de informações genômicas, bioquímicas e fisiológicas disponíveis em diversos bancos de dados e literatura especializada. Durante este processo, as enzimas FabZ e DesK, responsáveis por adicionar insaturações na cadeia carbono-carbono ao longo da síntese de ácidos graxos, foram identificadas no genoma, entretanto de forma truncada. O fluxoma da via metabólica foi definido com a descrição das rotas das principais reações, desde o metabólito de entrada, o Acetil-CoA, até o produto final, o ácido Hexadecenóico. A modelagem computacional foi feita com o uso do software MATLAB® juntamente com toolboxes e funções específicos para biologia de sistemas. A quantificação de metabólitos produzidos pela via foi realizada por meio do método baseado em restrições Análise de Balanço de Fluxos (ABF). Para avaliar a influência da expressão gênica na análise do fluxoma, o método ABF foi calculado usando os valores de log2FC obtidos na análise transcriptoma a 0ºC e 37ºC. A via de biossíntese de ácido graxos possui um total de 13 rotas identificadas pelo método Modo Elementar, quatro das quais apresentam caminhos para a produção de ácido hexadecenóico. A via reconstruída demonstrou a capacidade da E. antarcticum B7 em produzir moléculas de ácidos graxos. Sob a influência do transcriptoma, o fluxoma foi alterado, estimulando a produção de cadeia curta em ácidos graxos. Os modelos obtidos contribuem para um melhor entendimento da adaptação bacteriana a ambientes frios. / Mathematical modeling in silico based restrictions is an approach adopted by systems biology to analyze metabolic networks. The Gram-positive bacterium Exiguobacterium antarticum B7 is an extremophile organism able to survive in cold environments as glacial ice and permafrost. The ability of these microorganisms of adaptation to cold attracts great biotechnological interest. An important factor for the understanding of cold adaptation process is related to the chemical modification of fatty acids constituting the cell membrane of psicotrophic bacteria in order to maintain membrane fluidity to avoid freezing ofthe bacteria. In this work, the metabolic pathway of fatty acid biosynthesis of the bacterium E. antarticum B7 was rebuilt from its annotated genome. The software tools KEGG (Kyoto Encyclopedia of Genes and Genomes) and RAST (The Rapid Annotation Server) were used to generate a preliminary network model. The next step was to cure manually the genomic, biochemical and physiological informations available in different databases and specific literature. During this process, the FabZ and DesK enzymes responsible for adding carbon-carbon unsaturations in the fatty acid chain during synthesis have been identified in the genome, though in a truncated form. The fluxome metabolic pathway was defined, describing the routes of the main reactions since the first monomer, Acetyl-CoA, to the final product, the Hexadecenoic acid. A computational modeling was done using the software MATLAB® with toolboxes and specific tools for systems biology. The quantification of metabolites produced via was performed by the method constraint-based Flux Balance Analysis (FBA). To evaluate the influence of the gene expression in the fluxome analysis, the FBA method was also calculated using the log2FC values obtained in the transcriptome analysis at 0ºC and 37ºC. The fatty acid biosynthesis pathway showed a total of 13 elementary flux modes, four of which showed routes for the production of hexadecenoic acid. The reconstructed pathway demonstrated the capacity of E. antarcticum B7 to produce fatty acid molecules. Under the influence of the transcriptome, the fluxome was altered, promoting the production of short-chain fatty acids. The calculated models contributes to better understand the bacterial adaptation at cold environments.

This thesis examines determinacy and E-stability of economic policy in monetary union models. Monetary policy takes the form of either a contemporaneous or a forecast based interest rate rule, while fiscal policy follows a contemporaneous government spending rule. In the absence of asymmetries, the results from the closed economy literature on learning are retained. However, when introducing asymmetries into monetary union frameworks, the determinacy and E-stability conditions for economic policy differ from both the closed and open economy cases. We find that a monetary union with heterogeneous price rigidities is more likely to be determinate and E-stable. Specifically, the Taylor principle, a key stability condition for the closed economy, is now relaxed. Furthermore, an interest rate rule that stabilizes the terms of trade in addition to output and inflation, is more likely to induce determinacy and local stability under RLS learning. If monetary policy is sufficiently aggressive in stabilizing the terms of trade, then determinacy and E-stability of the union economy can be achieved without direct stabilization of output and inflation. A fiscal policy rule that supports demand for domestic goods following a shock to competitiveness, can destabilize the union economy regardless of the interest rate rule employed by the union central bank. In this case, determinacy and E-stability conditions have to be simultaneously and independently met by both fiscal and monetary policy for the union economy to be stable. When fiscal policy instead stabilizes domestic output gaps while monetary policy stabilizes union output and inflation, fiscal policy directly affects the stability of monetary policy. A contemporaneous monetary policy rule has to be more aggressive to satisfy the Taylor principle, the more aggressive fiscal policy is. On the other hand, when monetary policy is forward looking, an aggressive fiscal policy rule can help induce determinacy.

Dissertação (mestrado) - Fundação Getúlio Vargas, Curso de Mestrado em Administração Pública, Rio de Janeiro, 2010. / Bibliografia: 197-211 p. / Este estudo busca elaborar um sistema de avaliação do Programa Netuno de gestão da Marinha do Brasil, com base na literatura existente sobre o assunto. A pesquisa bibliográfica tem início com as origens da Administração Pública Brasileira, passa pelas teo / Made available in DSpace on 2018-02-14T18:06:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2010. Added 1 bitstream(s) on 2019-02-13T19:08:05Z : No. of bitstreams: 1 000016ce.pdf: 1637345 bytes, checksum: 68cf2595d3f9fc3b03b319c6568b4689 (MD5)

This thesis concerns the investigation of novel immunotherapies for cancer eradication. CpG therapy was used in order to target antigen-presenting cells (APCs), facilitating antigen presentation and activation of T cells. Blockade of the two major immune checkpoint regulators (CTLA-4 and PD-1) was also studied to ensure proper and sustained T cell activation. The therapies were investigated alone and compared to BCG, the standard immunotherapy in the clinic today for bladder cancer. In addition, CpG as well as BCG was combined with CTLA-4 or PD-1 blockade to examine if the combination could improve therapy. Single and combination strategies were assessed in an experimental bladder cancer model. In addition, one of the therapies (local aCTLA-4 administration) was evaluated in an experimental pancreatic cancer model. To be able to study the effects of CpG in humans, a human whole blood loop system has been used. This allowed us to dissect the potential interplay between CpG and complement. CpG was found to be superior to the conventional therapy, BCG, in our experimental model and T cells were required in order for effective therapy to occur. Used as a monotherapy, CTLA-4 blockade but not PD-1 blockade, prolonged survival of mice. When CTLA-4 or PD-1 blockade was combined with CpG, survival was enhanced and elevated levels of activated T cells were found in treated mice. In addition, Treg levels were decreased in the tumor area compared to tumors in control treated mice. CTLA-4 blockade was also effective when administrated locally, in proximity to the tumor. Compared to systemic CTLA-4 blockade, local administration gave less adverse events and sustained therapeutic success. When CpG was investigated in a human whole blood loop system it was found to tightly interact with complement proteins. This is an interesting finding which warrants further investigation into the role of TLRs in complement biology. Tumor therapy could be affected either negatively or positively by this interaction. The results presented herein are a foundation for incorporating these combination therapies into the clinic, specifically for bladder cancer but in a broader perspective, also for other solid tumors such as pancreatic cancer.