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La sénescence se caractérise par un arrêt en phase G1/S du cycle cellulaire et peut être induit par une variété de stress tels que des télomères trop courts, l’activation d’oncogène ou encore à cause de stress oxydatifs. Cette réponse cellulaire s’accompagne de profonds changements au niveau de l’expression génique et les ARN non codants sont d’importants acteurs de ceux-ci. Bien que cette catégorie d’ARN ait longtemps été considérée comme un sous-produit non fonctionnel de la transcription, on sait maintenant qu’ils sont impliqués dans une pléthore de fonctions essentielles à l’homéostasie de la cellule. Les microARN (miR), de petits ARN non codants d’une vingtaine de nucléotides, sont souvent diminués ou surexprimés dans les maladies, soulignant leurs rôles importants dans le développement de celles-ci. C’est le cas notamment de deux oncomirs, miR-19 et miR-155, qui s’accumulent de manière aberrante dans les cancers hématopoïétiques. En condition normale, STAT5A, qui est souvent dérégulé dans ces cancers, induit SOCS1 qui agit comme un frein sur cette voie de signalisation afin de prévenir une prolifération incontrôlée. Des travaux conduits dans notre laboratoire montrent que SOCS1 est aussi impliqué dans la sénescence, car il est capable d’activer p53, un important suppresseur tumoral. SOCS1 peut être ciblé par les deux oncomirs et nos résultats montrent qu’une inhibition de ces derniers à l’aide d’éponges artificielles favorisait l’accumulation d’un p53 actif. De plus, en intégrant le ribozyme à tête de marteau dans la conception des éponges, nous avons créé une nouvelle génération d’outils (éponges catalytiques) qui sont plus efficaces. Effectivement, l’utilisation de ces éponges contre miR-155 résultait en une diminution de la prolifération, de formation de colonie ainsi que de la migration de cellule de myélome multiple. En second lieu, nous nous sommes penchés sur l’étude d’éponges naturelles dans le contexte de la sénescence. Il existe en effet quelques exemples de lARNnc (Long Non-Coding RNA) qui peuvent agir de la sorte pour un miR donné. Nous pensons que c’est par ce mécanisme de régulation que miR-146a, un miR impliqué dans la réponse anti-inflammatoire, peut s’accumuler dans la sénescence induite par RAS sans toutefois sembler être pleinement actif. Effectivement, les cellules sénescentes sécrètent une variété de facteurs pro-inflammatoires. À l’aide d’une nouvelle technique nommée miR-CLIP, nous avons pu étudier l’interactome de miR-146a et avons identifié plusieurs lARNnc qui selon des outils de prédiction, semblent s’hybrider de manière extensive en région 3’ du miR. Ceci est requis pour l’initiation d’un TDMD (Target-Directed miR Degradation) et nous avons donc investigué la possibilité d’un tel évènement dans la régulation de miR-146a. Nos résultats montrent que la surexpression de XXBAC-B444P24.13 mène à une diminution des niveaux de miR-146a qui n’est pas due à une baisse de sa transcription. Bien que le premier article illustre les avantages d’une éponge catalytique artificielle, le second article suggère que cette stratégie pourrait déjà être en place dans les systèmes biologiques, et ce, de manière naturelle. En effet, une fois miR-146a lié à XXBAC-B444P24.13, ce dernier induirait la dégradation du miR par un TDMD. Ceci ouvre donc la porte au développement d’outils qui pourraient être plus performants à des niveaux d’expression plus bas. / Cellular senescence is characterized by a cell cycle arrest in the G1/S phase and can be induced by a variety of stresses which include telomere shortening, oncogene activation or oxidative stress. Its establishment is known to require changes in the genetic expression program and non-coding RNA play an important part in this phenomenon. For a long time, this RNA subtype was considered to only be a transcriptional byproduct, but we now know that they are involved in a plethora of functions which are essential to cell homeostasis. Various diseases display aberrant expression of microRNA (miR), small non-coding RNA of 18-22 nucleotides, suggesting they are involved in their development. Such is the case for miR-19 and miR-155, two oncomirs which are found to be overexpressed in hematopoietic cancers. In normal conditions, STAT5A, which is often found dysregulated in those cancers, induces SOCS1 which acts as a retro-inhibitor of this signaling pathway, preventing uncontrolled proliferation. Furthermore, our lab has shown that SOCS1 can also be involved in senescence by facilitating p53 activation. SOCS1 can be targeted by both oncomirs and our results show that artificial sponges, that inhibit miR-19 or miR-155’s functions, lead to the activation of p53. Also, we have incorporated the hammerhead ribozyme in the miR binding sites in the sponge, creating a sponge 2.0 (catalytic sponges). Expressing the latter in a multiple myeloma cell line (RPMI8226) resulted in less proliferation, colony formation and migration. Secondly, we aimed at studying natural sponges in the context of senescence. Indeed, there are quite a few examples of lncRNA (Long Non-Coding RNA) acting as a miRNA inhibitor by quenching them. We think that this mode of regulation could provide an explanation as to how an anti-inflammatory miR, miR-146a, can accumulate in senescence even though it is a pro-inflammatory response. Using a novel technique called miR-CLIP, we were able to study specifically miR-146a’s interactome and have found that it can interact with many lncRNAs. Interestingly, using computational tools, we noticed that miR-146a was predicted to interact with extensive 3’ end hybridization with a number of these lncRNA. This characteristic is known to be required to induce TDMD (Target-Directed miR Degradation). Indeed, when we overexpressed XXBAC-B444P24.13, miR-146a levels went down and this is not caused by a decrease in transcription of the miR. In the first part of this thesis, we show that artificial catalytic sponges have an advantage over a more “classical” design. This is further supported by the fact that this strategy seems to be employed in nature. Indeed, we might have uncovered a lncRNA that when bound to by miR-146a would lead to its degradation using TDMD. This could be taken advantage of in the development of new tools for miR inhibition that would be more powerful and could be potentially used at lower levels of expression.

Solution chemistry is a versatile and powerful tool in the synthesis of designed, complex nano-level high-tech materials. Normally, the technique is considered too expensive for large-scale production of complex multi-component ceramic materials. This thesis describes the expansion of the useful area of solution processing to multi-component bulk materials such as ZnO-based high-field varistors and WC–Co cutting tools, by developing novel techniques for solution-based coating of conventionally prepared metal and ceramic powders. The chemistry and microstructure development in the preparation of coatings, and the sintering of the coated powders to compacts, were studied in detail by SEM-EDS, TEM-EDS, XRD, IR-spectroscopy, dilatometry, TGA and DSC chemical analysis. ZnO powder with a ca 20 nm thick, homogeneous oxide coat of Bi–Sb–Ni–Co–Mn–Cr–Al oxide was prepared. After sintering to dense varistor bodies, much improved microstructures with much reduced ZnO-grain sizes were obtained. This shows that the oxides added as liquid sintering aid and grain-growth inhibitor become much more active when added homogeneously as a skin on the ZnO powder. After sintering of cobalt-coated WC, much improved micro-structures were obtained with a much more narrow WC grain-size distribution than that obtained from starting powders mixed by a conventional milling route. Coated powders also obviate the need for the extensive milling of WC and Co powders used in conventional mixing. The novel solution route was also applied to preparation of porous sponges and thin films on metal, glass and Al2O3 of sub 20 nm sized Co- or Ni-particles.

Este documento apresenta o Lyra, um novo esquema de derivação de chaves, baseado em esponjas criptográficas. O Lyra foi projetado para ser estritamente sequencial, fornecendo um nível elevado de segurança mesmo contra atacantes que utilizem múltiplos núcleos de processamento, como uma GPU ou FPGA. Ao mesmo tempo possui uma implementação simples em software e permite ao usuário legítimo ajustar o uso de memória e tempo de processamento de acordo com o nível de segurança desejado. O Lyra é, então, comparado ao scrypt, mostrando que esta proposta fornece um nível se segurança mais alto, além de superar suas deficiências. Caso o atacante deseje realizar um ataque utilizando pouca memória, o tempo de processamento do Lyra cresce exponencialmente, enquanto no scrypt este crescimento é apenas quadrático. Além disto, para o mesmo tempo de processamento, o Lyra permite uma utilização maior de memória, quando comparado ao scrypt, aumentando o custo de ataques de força bruta. / This document presents Lyra, a password-based key derivation scheme based on cryptographic sponges. Lyra was designed to be strictly sequential, providing strong security even against attackers that use multiple processing cores, such as FPGAs or GPUs. At the same time, it is very simple to implement in software and allows legitimate users to tune its memory and processing costs according to the desired level of security. We compare Lyra with scrypt, showing how this proposal provides a higher security level and overcomes limitations of scrypt. If the attacker wishes to perform a low-memory attack against the algorithm, the processing cost grwos expontetialy, while in scrypt, this growth is only quadratic. In addition, for an identical processing time, Lyra allows for a higher memory usage than its counterparts, further increasing the cost of brute force attacks.

Orientador: Maria Helena Robert / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Mecanica / Made available in DSpace on 2018-11-08T18:39:47Z (GMT). No. of bitstreams: 1 Silva_RenatoRafaelda_M.pdf: 15646556 bytes, checksum: 19ff8cd2fd9081ac7985bc44c94795f5 (MD5) Previous issue date: 2008 / Resumo: Este trabalho teve como objetivo estudar o processo de produção de esponjas metalicas por tixoconformacao em pré-formas removiveis e caracterizacao mecanico-metalurgica do produto, visando a compreensão da influencia de parametros de processo nas suas caracteristicas estruturais e propriedades. A liga empregada, AA2011, foi infiltrada sobre pre-formas de particulas de NaCl de granulometrias distintas, sinterizadas ou soltas. As esponjas metalicas produzidas foram caracterizadas quanto a sua arquitetura, propriedades fisicas (densidade real e relativa, condutividade e difusividade termicas) e mecanicas (tensão de compressão, modulo de Young, tensão no plato, deformação e energia de impacto absorvida); foi analisada a influencia do tipo de pre-forma utilizada para produção das esponjas, nestas caracteristicas e propriedades. Os resultados mostraram boa reprodutibilidade do processo, principalmente quando da utilização de pre-formas de partículas medias e grosseiras nao sinterizadas. A densidade da esponja e a espessura da parede celular apresentam tendencia a aumentar com o aumento das dimensões das celulas produzidas em preformas de particulas mais grosseiras. Todas as amostras produzidas apresentaram condutividade termica da ordem de 10x inferior e difusividade termica da ordem de 4x superior as do metal maciço. O comportamento das esponjas em compressão estatica ou dinamica se mostrou tipico de materiais celulares, com reduzidos valores de modulo de Young e de tensão de compressão, grande plato de deformação plástica sem acréscimo de tensão, e elevados valores de deformação total e energia absorvida no impacto / Abstract: The aim of this work was the analysis of the thixoforming process to produce metallic sponges and the mechanical and metallurgical characterization of the product, searching for better understanding the influence of processing parameters in the material structural characteristics and properties. The aluminium alloy AA2011 was infiltrated in the semi-solid state into performs of NaCl particles with different sizes and in sintered and non sintered conditions. Cellular products were characterized concerning internal architecture, physical properties (actual and relative densities, thermal conductivity and diffusivity) and mechanical properties (Young's modulus, compressive strength, plateau stress and absorbed impact energy); it was analyzed the influence of processing parameters on the properties and characteristics of the produced sponges. Results showed good reproducibility of the process, mainly when medium and coarse non sintered space holder particles were employed. Density of the product as well as cell wall thickness increases as space holder particles sizes increase. All the sponges produced showed low thermal conductivity (~10x inferior compared to the bulk material) and high thermal diffusivity (~3x superior compared to the bulk alloy). Results of static and dynamic compression tests showed typical cellular material behaviour in all cases, presenting low values for Young's modulus and compressive strength, a well defined plateau of plastic deformation, high plastic deformation and high capacity of energy absorption in impact events / Mestrado / Materiais e Processos de Fabricação / Mestre em Engenharia Mecânica

Este documento apresenta o Lyra, um novo esquema de derivação de chaves, baseado em esponjas criptográficas. O Lyra foi projetado para ser estritamente sequencial, fornecendo um nível elevado de segurança mesmo contra atacantes que utilizem múltiplos núcleos de processamento, como uma GPU ou FPGA. Ao mesmo tempo possui uma implementação simples em software e permite ao usuário legítimo ajustar o uso de memória e tempo de processamento de acordo com o nível de segurança desejado. O Lyra é, então, comparado ao scrypt, mostrando que esta proposta fornece um nível se segurança mais alto, além de superar suas deficiências. Caso o atacante deseje realizar um ataque utilizando pouca memória, o tempo de processamento do Lyra cresce exponencialmente, enquanto no scrypt este crescimento é apenas quadrático. Além disto, para o mesmo tempo de processamento, o Lyra permite uma utilização maior de memória, quando comparado ao scrypt, aumentando o custo de ataques de força bruta. / This document presents Lyra, a password-based key derivation scheme based on cryptographic sponges. Lyra was designed to be strictly sequential, providing strong security even against attackers that use multiple processing cores, such as FPGAs or GPUs. At the same time, it is very simple to implement in software and allows legitimate users to tune its memory and processing costs according to the desired level of security. We compare Lyra with scrypt, showing how this proposal provides a higher security level and overcomes limitations of scrypt. If the attacker wishes to perform a low-memory attack against the algorithm, the processing cost grwos expontetialy, while in scrypt, this growth is only quadratic. In addition, for an identical processing time, Lyra allows for a higher memory usage than its counterparts, further increasing the cost of brute force attacks.

<p>Solution chemistry is a versatile and powerful tool in the synthesis of designed, complex nano-level high-tech materials. Normally, the technique is considered too expensive for large-scale production of complex multi-component ceramic materials. This thesis describes the expansion of the useful area of solution processing to multi-component bulk materials such as ZnO-based high-field varistors and WC–Co cutting tools, by developing novel techniques for solution-based coating of conventionally prepared metal and ceramic powders. The chemistry and microstructure development in the preparation of coatings, and the sintering of the coated powders to compacts, were studied in detail by SEM-EDS, TEM-EDS, XRD, IR-spectroscopy, dilatometry, TGA and DSC chemical analysis. </p><p>ZnO powder with a ca 20 nm thick, homogeneous oxide coat of Bi–Sb–Ni–Co–Mn–Cr–Al oxide was prepared. After sintering to dense varistor bodies, much improved microstructures with much reduced ZnO-grain sizes were obtained. This shows that the oxides added as liquid sintering aid and grain-growth inhibitor become much more active when added homogeneously as a skin on the ZnO powder.</p><p>After sintering of cobalt-coated WC, much improved micro-structures were obtained with a much more narrow WC grain-size distribution than that obtained from starting powders mixed by a conventional milling route. Coated powders also obviate the need for the extensive milling of WC and Co powders used in conventional mixing.</p><p>The novel solution route was also applied to preparation of porous sponges and thin films on metal, glass and Al₂O₃ of sub 20 nm sized Co- or Ni-particles. </p>

Chemoreception plays an important role in predator-prey interactions and feeding dynamics. While the chemoreception of attractant or pleasant tasting compounds has been well studied, aversive chemoreceptive signaling has been difficult to investigate behaviorally in an ecological context because these interactions are species- and context- specific and deterrent compounds vary among prey. Using the coral reef system, this thesis explores on a molecular level the deterrent mechanism underlying detection by fish predators of an aversive compound, in order to gain a greater understanding of predator-prey interactions in this community. Like other organisms that are sessile or slow-moving, marine sponges have special mechanisms for defense from predation, commonly containing aversive-tasting compounds that defend these organisms from predation. To this end, we sought to identify and characterize a fish chemoreceptor that detects one or more of these compounds. We isolated a single cDNA clone encoding RAMP-like triterpene glycoside receptor (RL-TGR), a novel co-receptor involved in the signaling of triterpene glycosides. This co-receptor appears to be structurally and functionally related to receptor activity-modifying proteins (RAMPs), a family of co-receptors that physically associate with and modify the activity of G protein-coupled receptors (GPCRs). Expression in Xenopus oocytes showed that it responds to triterpene glycosides in a receptor-mediated manner and requires co-expression of a GPCR to enable signaling in oocytes; both of these receptors may be components of a larger signaling complex. A 40 bp portion of the gene is conserved across multiple fish species, but is not found in any other organism with a sequenced genome, suggesting that the expression of this receptor is limited to fish species. RL-TGR is the first identified gene encoding a co-receptor that responds to a chemical defense. This finding may lead the way for the identification of many other receptors that mediate chemical defense signaling in both marine and terrestrial environments, as this protein has the potential to represent the first of an entire family of co-receptors that respond to aversive compounds.

Orientador: Lara Durães Sette / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T06:41:16Z (GMT). No. of bitstreams: 1 Passarini_MichelRodrigoZambrano_D.pdf: 6997453 bytes, checksum: 0a905c32bd8d912eb875c88cff54de8e (MD5) Previous issue date: 2012 / Resumo: O oceano representa um habitat promissor na busca por novos micro-organismos, os quais podem apresentar capacidade de produzir enzimas de interesse industrial diferentes das produzidas por seus parceiros terrestres. Neste contexto, duas amostras da esponja marinha Dragmacidon reticulatum foram coletadas no litoral Norte do Estado de São Paulo, objetivando a caracterização da diversidade fúngica por métodos dependentes e independentes de cultivo, bem como a avaliação da produção, expressão da enzima e caracterização do gene da lacase. Com relação à parcela cultivada das amostras, 108 fungos filamentosos foram isolados. Destes, 64 ribotipos distintos foram submetidos aos experimentos de taxonomia polifásica e aos relacionados com a lacase. Análises macro- e microscópicas, moleculares (genes ribossomais ITS/28S) e pela técnica de MALDI TOF ICMS, resultaram na caracterização de 38 isolados distribuídos em 23 gêneros pertencentes ao Filo Ascomycota e um ao Filo Zygomycota. Este foram posteriormente depositados na Coleção Brasileira de Micro-organismos de Ambiente e Indústria (CBMAI). Dentre os isolados obtidos, uma potencial espécie nova de Penicillium foi identificada. Os resultados da triagem enzimática permitiram a seleção de dois isolados identificados como Nigrospora sp. CBMAI 1328 (0,30 U L-1) e Arthorpyrenia sp. CBMAI 1330 (0,40 U L-1), os quais foram submetidos à uma nova avaliação da atividade e expressão (RT-PCR) com indução de íons cobre e caracterização do genes da lacase. A adição de íons cobre na concentração de 5 mM, proporcionou aumento das atividades enzimáticas dos isolados CBMAI 1328 e CBMAI 1330 em 3,9X (25,2 U L-1) e 1,2X (9,0 U L-1) após 120 h, respectivamente. Os resultados da expressão dos genes da lacase para o isolado CBMAI 1328 foram os mesmos encontrados pela indução com cobre (maior expressão em 5 mM após 120 h), entretanto para o isolado CBMAI 1330, a maior expressão foi após 96 h sem adição de cobre. Os resultados da caracterização dos genes da lacase revelaram a possivel existência de 3 novos putativos genes de lacases marinhas. Com relação à parcela não cultivada, foi possível a identificação de 7 gêneros e de um fungo não cultivado (uncultured fungi) do Filo Ascomycota bem como 3 gêneros e um fungo não cultivado (uncultured fungi) do Filo Basidiomycota a partir da técnica de DGGE e do sequenciamento direto do gene RNAr ITS e do gene 18S. Em ambas as abordagens utilizadas, a maior diversidade foi encontrada na amostra DR9. Os dados derivados do presente trabalho, ressaltam a importância em se utilizar a taxonomia polifásica, bem como empregar metodologias dependente e independente de cultivo (em paralelo) para um melhor e maior conhecimento da real diversidade em amostras ambientais. Estes resultados ampliam o conhecimento dos fungos filamentosos recuperados de esponjas marinhas e demonstram o potencial biotecnológico destes micro-organismos / Abstract: The ocean represents a promissing habitat in the search for new microorganisms, which may have the ability to produce enzymes of industrial interests different from that produced by their terrestrial counterparts. In this context, two samples of the marine sponge Dragmacidon reticulatum were collected on northern coast of São Paulo State, aiming at the characterization of fungal diversity by cultureddependent and -independent approaches as well as the evaluation of the production, characterization and expression of laccase enzyme gene. Regarding the cultivated portion of the samples, around 108 filamentous fungi were isolated, belonging to 64 different taxonomic groups (ribotypes), which were subjected to enzymatic activity assays. Polyphasic taxonomy approaches (macro- and microscopic, molecular - ITS/28S ribosomal genes - and MALDI TOF ICMS analyses) resulted in the characterization of 38 isolates distributed in 23 genera belonging to the phylum Ascomycota and one to the phylum Zygomycota, which were deposited in the Brazilian Collection of Micro-organisms from Environment and Industry (CBMAI). Among the isolates recovered one possible new species of Penicillium was identified. Enzymatic screening allowed the selection of two isolates identified as Nigrospora sp. CBMAI 1328 (0,30 U L-1) and Arthorpyrenia sp. CBMAI 1330 (0,40 U L-1), which were subjected to a new activity evaluation and expression (RT-PCR) with the induction of copper ions and the laccase genes characterization. The addition of copper ions in a concentration of 5 mM resulted in an increase in the enzymatic activities of CBMAI 1328 and CBMAI 1330 strains in 3,9X (25,2 U L-1) and 1,2X (9,0 U L-1) after 120h, respectively. The results of the expression of the laccase genes for CBMAI 1328 strain were the same found by induction with copper (expression increased in 5 mM after 120 h), however for CBMAI 1330 the higher expression was after 96 h without addition of copper. Results of laccase genes characterization revealed the existence of three possible putative new marine laccase genes. Regarding to the uncultured portion of the samples, from the DGGE and direct sequencing of the ITS rRNA gene and 18S gene approaches, was possible to identify seven species of fungi and one uncultured fungus from phylum Ascomycota and three species and one uncultured fungus from phylum Basidiomycota. For both approaches used the greatest diversity was achieved in the DR9 sample. Data derived from the present work highlight the importance of using polyphasic taxonomy as well as applying culturedependent and culture-independent methodologies (in parallel) in order to have a better and greater knowledge of the real diversity in environmental sample. These results expand the knowledge of fungi recovery from marine sponges and demonstrate the biotechnological potential of these micro-organisms / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular