Orientador: Jose Barreto Campello Carvalheira / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T01:57:33Z (GMT). No. of bitstreams: 1 Fernandes_MariaFernandadeAndrade_M.pdf: 2919034 bytes, checksum: c3f7796b55fb5ad53b9eaf2ee58536d5 (MD5) Previous issue date: 2007 / Resumo: As proteínas AMPK e mTOR são as principais moduladoras do balanço energético intracelular, e exercem influência decisiva sobre a ação hipotalâmica da leptina. O exercício agudo, através da produção de IL-6, está associado ao aumento da sensibilidade à ação anorexigênica da leptina. Visando investigar a possível influência das vias AMPK e mTOR, neste aumento da sensibilidade hipotalâmica à leptina induzido pelo exercício agudo, ratos Wistar foram submetidos à natação e posteriormente receberam injeção intracerebroventricular de IL-6, ativadores e/ou inibidores da AMPK e mTOR. A IL-6 reduziu a ingestão alimentar dos animais não exercitados; no entanto, o pré-tratamento com ativador da AMPK ou inibidor da mTOR bloqueou esta ação da IL-6. Ativadores da AMPK aumentaram a ingestão alimentar de forma mais significativa nos animais não exercitados. Inibidores da AMPK reduziram a ingestão mais expressivamente nos animais exercitados. A injeção central de leptina reduziu a ingestão de ratos exercitados mais expressivamente do que foi observado nos animais controle. Tanto o pré-tratamento com inibidor da IL-6, como com ativador da AMPK ou inibidor da mTOR, reverteram esta ação da leptina. O exercício também está associado à redução da fosforilação da via da AMPK e à maior fosforilação da via da mTOR, no hipotálamo. A da resposta das vias em questão ao estímulo com leptina provavelmente seja um dos principais determinantes da modulação do set point hipotalâmico pelo exercício agudo / Abstract: AMP-activated protein kinase (AMPK) and mammalian Target of Rapamycin (mTOR) are key regulators of cellular energy balance and of the effects of leptin on food intake. Acute exerci se is associated with increased sensitivity to the effects of leptin on food intake in an IL-6-dependent manner. To determine whether exerci se ameliorates the AMPK and mTOR response to leptin in the hypothalamus in an IL-6-dependent manner, rats performed two 3-h exercise bouts, separated by one 45-min rest períod. Intracerebroventrícular IL-6 infusion reduced food intake and pretreatment with AMPK activators and mTOR inhibitors prevented IL-6-induced anorexia. Activators of AMPK increased food intake in control rats to a greater extent than that observed in exercised ones, whereas inhibitors of AMPK had the opposite effect. Exercise was associated with both reduced phosphorylation of the AMPK/ ACC signaling pathway and increased phosphorylation of proteins involved in mTOR signal transduction Ín the hypothalamus. The regulatory role of IL-6 in mediating the modulation in AMPK and mTOR pathways in the hypothalamus was also investigated. Treatment with leptin reduced food intake in exercised rats that were pretreated with vehicle, although no increase in sensitivity to leptin-induced anorexia after pretreatment with anti-IL6 antibody, AICAR or Rapamycin was detected. Improved responses of AMPK and mTOR to leptin may contribute to the appetite suppressive actions of exercise / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

Este trabalho apresenta um estudo de Qualidade de Energia Elétrica (QEE) no sistema elétrico industrial de uma concessionária geradora de energia elétrica do Brasil. O estudo teve enfoque na distorção harmônica gerada nos serviços auxiliares da usina termoelétrica. Para tanto, foi necessária a simulação e análise da planta da termoelétrica, com atenção nos serviços auxiliares pertencentes a ela, devido aos altos níveis de distorções harmônicas registrados. A simulação foi feita com o programa ATP (Alternative Transients Program) no domínio do tempo, através de sua interface gráfica, o ATP Draw. Nele foram modelados os principais componentes que pertencem ao Sistema Elétrico de Potência (SEP) em questão, como geradores, transformadores, linhas e cargas não lineares. Com a modelagem do SEP da termoelétrica, é possível determinar os níveis da distorção harmônica em diversos locais da planta, e aprofundar o estudo para que seja possível fazer a mitigação desses distúrbios. A diminuição da distorção harmônica junto ao aumento do fator de potência permite um melhor desempenho da usina, aumentando a faturamento da empresa através do aumento da energia entregue ao sistema elétrico. / This dissertation presents a study of Power Quality in an electrical system of a thermoelectric power plant in Brazil. The study focused on the harmonic distortion generated in the auxiliary services of the plant. Therefore, it was necessary to simulate and analyze the thermal plant, with attention to auxiliary services belonging to it, due to high levels of harmonic distortion registered. The simulation was performed using the ATP (Alternative Transients Program) in the time domain through its interface, the ATP Draw. It were modeled the main components that belong to the Power System in question, such as generators, transformers, lines and non-linear loads. With the modeling of the thermoelectric plant, is possible to determine the levels of harmonic distortion at various locations within the plant. The reduction of harmonic distortion along with the increase of power factor allows a better plant performance, increasing company revenue by increasing the energy delivered to the electrical system.

Submitted by Rafael Santana (rafael.silvasantana@ufpe.br) on 2018-02-06T19:46:42Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) DissertacaoSuellen_sem_assinatura.pdf: 3437351 bytes, checksum: 69795cdbb0c8879d128ec5de560c03d2 (MD5) / Made available in DSpace on 2018-02-06T19:46:42Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) DissertacaoSuellen_sem_assinatura.pdf: 3437351 bytes, checksum: 69795cdbb0c8879d128ec5de560c03d2 (MD5) Previous issue date: 2016-08-29 / CNPQ / Atualmente, a expansão da demanda energética acontece por dois motivos principais: o aumento da população mundial e do consumo energético per capita. Um dos desafios no contexto de distribuição de energia está relacionado a regulação dos níveis de tensão das barras de transmissão no sistema de distribuição de energia. Para correção de comportamentos indesejáveis (e.g., flutuações nos níveis de tensão), reatores naturalmente saturados (RNSs) são empregados na compensação de reativos ajustando os níveis de tensão do sistema elétrico de potência. Além da utilização de protótipos para validação de cenários do sistema elétrico, modelos computacionais de RNSs podem facilitar a modelagem de reatores utilizando ferramentas de softwares. Tais modelos auxiliam o projeto destes equipamentos e diminuem significativamente seu custo de prototipação. Este trabalho apresenta um esquema de modelagem e uma biblioteca de simulação de RNSs utilizando modelos de circuito magnético unificado (UMEC) na linguagem MODELS do software Alternative Transient Program (ATP). A biblioteca implementa características comuns a diferentes tipos de RNSs tornando simples a criação de novos modelos sem a necessidade de reescrita de todo o código para cada componente. Dessa forma, engenheiros podem simular o comportamento de RNSs com diferentes configurações de uma maneira simples e efetiva. Neste trabalho, diversos estudos de caso são mostrados com o intuito de se apresentar a viabilidade da solução apresentada. / Currently, the energetic demand increase occurs due to two main reasons: the rise of world population and expansion of per-capita consumption. A key challenge in the context of power distribution is related to regulating the voltage levels of transmission bars in power distribution systems. In order to fix undesirable behavior (e.g., fluctuations in voltage levels), naturally saturated reactors (RNSs) are employed in the compensation of reactive power and adjusting the electrical system voltage levels. The complexity and high cost of creating actual prototypes for validating electrical system scenarios requires the creation of computer models of RNSs. Such models help the design of equipment and significantly reduce their cost of prototyping. This paper presents a modeling scheme and an RNS simulation library using Unified Magnetic Equivalent Circuit (UMEC) in MODELS language of Alternative Transient Program (ATP) software. The library implements common features for different types of RNSs facilitating the creation of new RNSs without the need write the all the source code from scratch for each component. Thus, engineers can simulate the behavior of RNSS with different configurations in a more simple and effective way. In this work, several case studies are shown to demonstrate the viability of the proposed solution.

Submitted by Luanna Matias (lua_matias@yahoo.com.br) on 2015-03-06T16:58:22Z No. of bitstreams: 2 Tese - Sara Fernandes Soares - 2012.pdf: 2842867 bytes, checksum: dff92fa87ddeabaa7f16510a5ac58f11 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luanna Matias (lua_matias@yahoo.com.br) on 2015-03-06T17:07:16Z (GMT) No. of bitstreams: 2 Tese - Sara Fernandes Soares - 2012.pdf: 2842867 bytes, checksum: dff92fa87ddeabaa7f16510a5ac58f11 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-03-06T17:07:16Z (GMT). No. of bitstreams: 2 Tese - Sara Fernandes Soares - 2012.pdf: 2842867 bytes, checksum: dff92fa87ddeabaa7f16510a5ac58f11 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2012-02-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Rhipicephalus sanguineus is an ectoparasite of domestic dogs which can also be found in other mammals, including humans. It has high medical and veterinary importance, given that it can transmit pathogens that cause diseases such as Rocky Mountain spotted fever and Boutonneuse fever in humans and babesiosis and ehrlichiosis in dogs. The main control method to this tick is the use of chemical acaricides which, over the years, has selected resistant tick populations to these products. To develop strategies to control this tick, it is necessary to know its ecology. Electrophysiological techniques are important tools in the study of substances that interfere with the behavior of animals. In this study, the existence of gustatory sensilla on the chelicerae of R. sanguineus was investigated by scanning electron microscopy (SEM). Also, the electrophysiological responses of the neurons present in this sensilla in nonfed ticks to substances with the potential to act as phagostimulant, such as salts (KCl and NaCl), sugars (glucose, sucrose and fructose), the nucleotide adenosine triphosphate (ATP), the tripeptide reduced glutathione (GSH) and two purine (guanine and hypoxanthine) were assessed. Phytoecdysteroids (PES), compounds analogous to ecdysteroids, known as the molting hormones in arthropods, were also tested in electrophysiology. The PES ecdysone (E), 20-hidroxyecdysone (20E), ponasterone A (PonA), makisterone A (MakA), inokosterone (Inok) and Pterosterone (Pte) were tested on nonfed and fed ticks. To evaluate the influence the PEs identified as active in electrophysiology on feeding behavior of R. sanguineus, attachment bioassays in vivo were proceeded. The images obtained with SEM revealed the existence of a pore in the inner digit of the chelicerae, involved in taste perception in these ticks. The results obtained in electrophysiology showed strong activity of R. sanguineus cheliceral neurons to glucose and GSH, at concentrations above 10-4 M, to ATP from 10-2 M, and salts from 10-1 M. The action potentials observed in response to ATP at all used concentrations (from 10-6 M to 10-2 M), and to KCl at 1 M were from different neurons, while the action potentials to the other potentially phagostimulant stimuli were from a single neuron. Considering the responses to PEs in nonfed ticks, MakA and Pte triggered action potentials frequencies greater than the negative control, with detection thresholds of 10-6 M and 10-12 M, respectively. The action potentials amplitudes for these substances as well as for 20E and PonA were higher than those for the control, indicating the activity of a different neuron from that observed for the negative control. In fed ticks, only Pte at 10-4 M remained active. In the behavior assays, there was no difference in attachment between PEs and the control and no interference in the biological parameters was observed. The results obtained in this study showed the ability of R. sanguineus to detect in their cheliceral taste sensilla substances with different natures, as potential phagostimulants and PEs. Further studies in this area are needed to elucidate the role of these substances in the chemical ecology of these ticks. / Rhipicephalus sanguineus é um ectoparasita de cães domésticos, também encontrado em outros mamíferos, inclusive no homem. Tem elevada importância nas medicinas humana e veterinária, podendo transmitir agentes patogênicos causadores de doenças, como as febres botonosa e maculosa em humanos e a babesiose e a erliquiose em cães. Seu controle é feito com o uso de acaricidas químicos, o que, ao longo do tempo, selecionou populações deste carrapato resistentes a estes produtos. Para a elaboração de estratégias de controle deste carrapato é necessário conhecer sua ecologia. Técnicas eletrofisiológicas são ferramentas importantes na identificação de substâncias que interferem no comportamento de animais. Neste trabalho foi investigada a existência de sensilas gustativas nas quelíceras de R. sanguineus por meio da microscopia eletrônica de varredura (MEV). Também se avaliou a resposta eletrofisiológica dos neurônios destas sensilas, em carrapatos não alimentados, a substâncias com potencial para efeito fagoestimulante, tais como sais (KCl e NaCl), açúcares (glicose, sacarose e frutose), o nucleotídeo trifosfato de adenosina (ATP) e o tripeptídeo glutationa reduzida (GSH), assim como purinas (guanina e hipoxantina). Fitoecdisteróides (FES), compostos análogos aos ecdisteróides, conhecidos como hormônios da muda em artrópodes, foram igualmente avaliados pela eletrofisiologia. Foram empregados carrapatos antes e após a alimentação e os seguintes FEs: ecdisona (E), 20-hidroxiecdisona (20E), ponasterona A (PonA), makisterona A (MakA), inokosterona (Inok) e pterosterona (Pte). Para avaliar a interferência, no comportamento de alimentação de R. sanguineus, dos FEs que foram identificados ativos na eletrofisiologia, foram empregados testes de fixação in vivo. As imagens obtidas com a MEV evidenciaram a existência de um poro no dígito interno das quelíceras, envolvido na percepção gustativa, nesses carrapatos. Os resultados obtidos com a eletrofisiologia revelaram forte atividade de neurônios quelicerais de R. sanguineus à glicose e à GSH, em concentrações acima de 10-4 M, ao ATP, a partir de 10-2 M, e aos sais a partir de 10-1 M. Os potenciais de ação observados em resposta ao estímulo com ATP, em todas as concentrações empregadas (de 10-6 M a 10-2 M), e ao KCl a 1 M foram oriundos de diferentes neurônios, enquanto que aos demais estímulos potencialmente fagoestimulantes foram provenientes de um único neurônio. Quanto aos FEs, em carrapatos não alimentados, MakA e Pte desencadearam frequências de potenciais de ação superiores ao controle negativo, com limiares de detecção de 10-6 M e 10-12 M, respectivamente. As amplitudes dos potenciais de ação para estas substâncias bem como para 20E e PonA foram maiores que as do controle, indicando a atividade de um neurônio diferente do observado para o controle negativo. Nos carrapatos após alimentação, somente Pte à 10-4M permaneceu ativa. Nos testes comportamentais, não houve diferença de fixação entre os tratamentos com FEs e com o controle negativo e nem interferência do FEs nos parâmetros biológicos avaliados. Os resultados obtidos no presente estudo evidenciaram a capacidade de R. sanguineus em detectar, em suas sensilas gustativas quelicerais, substâncias de diferentes naturezas, como potenciais fagoestimulantes e FEs. Mais estudos nesta área são necessários visando esclarecer o papel destas substâncias na ecologia química destes carrapatos.

A sinalização purinérgica tem sido demonstrada como um um importante modulador dediversos processos fisiológicos e fisiopatológicos. Dentre os receptores purinérgicos, o receptor P2X7 distingui-se por requerir altas concentrações de ATP em sua ativação. As demonstrações prévias de que a glândula pineal responde a diferentes estímulos purinérgicose a altas concentrações de ATP sugere um papel para os receptores P2X7 nesta glândula, embora sua expressão e função não estivesse estabelecida. O objetivo deste trabalho, portanto, foi caracterizar funcionalmente o receptor P2X7 na glândula pineal de ratos. Os resultados obtidos demonstram, pela primeira vez, a expressão gênica e proteica do receptor P2X7 na glândula pineal. Os efeitos da ativação destes receptores levam a uma inibição nos níveis de melatonina induzida por isoprenalina por um mecanismo independente da via do fator de transcrição NF-kB e da fosfolipase C. Além disso, a estimulação destes receptores inibiu a síntese de TNF induzida por LPS, resultado este semelhante ao observado na presença do pré-tratamento com antagonistas do receptor P2X7. Estes dados confirmam a presença de receptores P2X7 na glândula pineal e reiteram o relevante papel da estimulação purinérgica a sobre a síntese de melatonina e sobre a capacidade da pineal em responder a PAMPs, como o LPS / Purinergic signalling has been demonstrated as an important modulator ofseveral physiological and pathophysiological processes. Among the purinergic receptors, the activation of P2X7 receptor requireshigh concentrations of ATP. The previous demonstration that the pineal gland is responsive to different purinergic stimulus and to high concentrations of ATPsuggests a role for P2X7, although its expression and function remained unclear. The aim of this study was to functionally characterize the P2X7 receptor in the rat pineal gland.The data showedthe P2X7 receptor mRNA and protein expression in the pineal gland. The effect of its activation leads toan inhibition of melatonin content induced by isoprenaline through an independent NF-kB and PLC pathways. Furthermore, the P2X7 receptor activation inhibits the LPS-induced TNF synthesis, a similar result observed in the presence of the pre-treatment with P2X7 receptors antagonists. These data demonstrate the presence of P2X7 receptors in the rat pineal gland and confirm the relevant role of the purinergic stimulation to the pineal melatonin synthesis and responsiveness to PAMPs such as LPS

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-09-20T14:10:45Z No. of bitstreams: 1 adbeeldelimasantos.pdf: 1016205 bytes, checksum: 9523680dcc32c8167da738acfd5b67d1 (MD5) / Approved for entry into archive by Diamantino Mayra (mayra.diamantino@ufjf.edu.br) on 2016-09-26T20:24:29Z (GMT) No. of bitstreams: 1 adbeeldelimasantos.pdf: 1016205 bytes, checksum: 9523680dcc32c8167da738acfd5b67d1 (MD5) / Made available in DSpace on 2016-09-26T20:24:29Z (GMT). No. of bitstreams: 1 adbeeldelimasantos.pdf: 1016205 bytes, checksum: 9523680dcc32c8167da738acfd5b67d1 (MD5) Previous issue date: 2010-03-03 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A segurança e a viabilidade econômica da transformação do leite em seus diversos derivados dependem fundamentalmente da correta aplicação das técnicas de higienização. Sendo assim, o objetivo principal deste trabalho foi avaliar as condições higiênico-sanitárias do ambiente e das superfícies de processamento de uma unidade produtora de queijo Canastra por meio da técnica de ATPBioluminescência e propor um procedimento de higienização que contribua para a segurança e melhoria desse sistema de produção. Também foi avaliada a água utilizada na obtenção do leite e no interior da queijaria. No período mais susceptível às contaminações, ou seja, no verão, foram escolhidos 12 pontos de amostragem pertencentes ao fluxograma de produção do queijo Minas artesanal, além da água utilizada na sala de ordenha e da água utilizada no interior da queijaria. Esses pontos foram avaliados pelos métodos tradicional (referência) e ATPBioluminescência. Após isso, foi sugerido um novo procedimento de limpeza e sanitização para esses pontos, que foram avaliados ao longo das quatro estações também por ambos os métodos. Houve diferença (p<0,05) entre os resultados obtidos para as avaliações realizadas antes e após a aplicação do procedimento proposto para a maioria dos pontos amostrados. A microestrutura das superfícies de equipamentos e utensílios apresentaram uma forte interferência, pois os resultados para os pontos de amostragem mesa de enformagem e prensagem (MEP), prateleira de maturação (PM) e formas (F) não diferiram (p>0,05) independentemente do procedimento de higienização e das estações do ano. O método de ATP-Bioluminescência não apresentou concordância com o método de tradicional quanto à classificação das condições higiênicas das superfícies avaliadas. Isso deve muito possivelmente à presença de ATP de origem não microbiana oriunda de resíduos que não foram completamente eliminados na etapa de limpeza. Os resultados encontrados indicam que o método de ATPBioluminescência deve ser empregado como indicador da presença de material biológico na superfície ou água, não substituindo o método tradicional. / The food safety and the economical viability of milk processing depend on appropriated higyenization techniques. The main goal of this work was to evaluate higienics conditions of food processing environment and surfaces of a producer farm of Canastra cheese by using ATP- Bioluminescence. In addition it was also proposed a higyenization procedure to improve safety and quality of manufacture proceeding. It was also evaluated the water quality used in milking and during cheese production. During summer, which is more probability to microbial contamination, 12 points of Canastra cheese production flow were chosen. Such samples were analyzed by traditional and ATP- Bioluminescence techniques during all seasons. It was suggested a new procedure of cleaning and sanitation for both traditional and ATP- Bioluminescence methods. There was diference (p<0.05) between the results obtained before and after the proposed higyenization procedure for the most samples evaluated. The microstructure of equipments and utensils surfaces showed a strong influence in results. The results for forming and pressing table, ripening rack and forms did not differ (p>0.05) independent of higyenization procedure and year seasons. ATP- Bioluminescence did not agree with the reference method in relation to the classification of higyenic conditions of evaluated surfaces. Probably, the presence of non-microbial ATP from food residues contributed to increasing the ATP level. The results found in the present work showed that ATP- Bioluminescence technique should be used as an indicator of biological material in water and/or surface; and it does not replace the traditional method.

Lignin is a key component of plant secondary cell walls, providing strength to the plant and allowing water transport. Lignin is a polymer of monolignols that are synthesized in the cell and transported into the cellulose rich cell wall. The primary goal of this thesis is to understand the mechanism(s) of monolignol deposition during xylogenesis. The currently accepted theory is that monolignols are exported by Golgi-mediated vesicle delivery to the secondary cell wall. When this theory was re-examined using cryofixed developing pine, quantitative autoradiography showed that monolignols did not accumulate in Golgi but were rapidly translocated from cytosol to cell wall. This suggests alternative mechanisms, such as membrane transporters, work in monolignol export. ATP binding cassette (ABC) transporters were chosen because they transport other secondary metabolites and some ABC transporter encoding genes are highly expressed in lignifying cells. Four candidate ABC transporters were selected in Arabidopsis (ABCB11, ABCB14, ABCB15 from the ABCB/MDR subfamily and ABCG33 from the ABCG/PDR subfamily) and shown to have overlapping, high vasculature expression patterns. Mutants with T-DNA insertions in single ABC transporter genes had no change in lignification of inflorescence stems. However, a reduced polar auxin transport phenotype was detected in mutants of ABCB11, ABCB14 and ABCB15. An additional approach was the use of inhibitors of ABC transporters. A new assay, which was developed to quantify lignification in primary xylem of Arabidopsis roots, demonstrated that ABC inhibitors did not change lignin deposition. Monolignols are exported and polymerized in the polysaccharide matrix of the cell wall, which includes hemicelluloses that may organize monolignols during polymerization. Since diverse lignified cell types are enriched in either G- or S-lignin, I hypothesized that this pattern could reflect different hemicellulose distributions, which was examined using antibody labeling of xylans or mannans in hybrid poplar xylem. While xylans were generally distributed in all secondary cell walls, mannans were enriched in fibers but not in the ray and vessel walls. In summary, during secondary cell wall deposition, monolignols are exported by unknown transporter(s) rather than Golgi vesicles. In developing poplar wood, the monolignols are deposited into diverse hemicellulose domains in different cell types. / Science, Faculty of / Botany, Department of / Graduate

Les DYRKs (dual specificity, tyrosine phosphorylation regulated Kinases) et CLKs (cdc2-like kinases) sont deux familles de kinases du groupe CMGC. Elles sont impliquées dans le développement de la maladie d'Alzheimer et de la trisomie 21. Nous présentons ici une optimisation et une caractérisation biologique détaillée des Leucettines, une famille d’inhibiteurs pharmacologiques de DYRKs/CLKs dérivés de la Leucettamine B, un alcaloïde extrait d'une éponge marine. Nous avons étudié la relation structure/activité de cette classe d'inhibiteurs sur un ensemble de réponses biologiques. Afin d'étudier les cibles potentielles de ces inhibiteurs, nous avons mis en œuvre une méthode de chromatographie d’affinité. La sélectivité de la Leucettine L41, sélectionnée comme représentative des Leucettines, a été étudiée par des essais d'activité et d'interaction de kinases recombinantes in vitro, et des tests de chromatographie d'affinité (Leucettines immobilisées sur billes d'agarose, compétition sur billes d’inhibiteurs non sélectifs). Des approches transcriptomiques et protéomiques ont été utilisées afin de mieux comprendre le mécanisme d'action cellulaire de la Leucettine L41. Ces approches ont confirmé la sélectivité de la Leucettine L41 pour DYRKs et CLKs mais aussi révélé l’existence de cibles secondaires intéressantes. La Leucettine L41 module l'épissage alternatif de pré-ARNm. Elle présente des propriétés neuroprotectrices vis-à-vis de la mort cellulaire induite par le glutamate. Les Leucettines méritent un développement en tant qu'agents thérapeutiques potentiels pour le traitement de la maladie d'Alzheimer et de la trisomie 21. / DYRKs (dual specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases) are two families of kinases belonging to the CMGC group. They are involved in the development of Alzheimer's disease and Down syndrome. We here present the optimization and a detailed biological characterization of Leucettines, a family of pharmacological inhibitors of DYRKs/CLKs derived from Leucettamine B, an alkaloid produced by a marine sponge. We studied the structure/activity relationship of this class of inhibitors on a set of biological responses. To investigate potential targets of these inhibitors, we implemented an affinity chromatography method. The selectivity of Leucettine L41, selected as a representative Leucettine, was studied by in vitro activity and interaction assays of recombinant kinases and affinity chromatography approaches (Leucettines immobilized on agarose beads, competition on non-selective inhibitors). Transcriptomics and proteomics approaches were used to better understand the cellular mechanism of action of Leucettine L41. These approaches confirmed the selectivity of Leucettine L41 for DYRKs and CLKs but also revealed the existence of interesting secondary targets. Leucettine L41 modulates alternative splicing of pre-mRNAs. It displays neuroprotective properties towards glutamate-induced cell death. Leucettines deserve further development as potential therapeutic agents for the treatment of Alzheimer's disease and Down syndrome.

Malaria, caused by an intracellular Plasmodium parasite, remains a devastating disease, having claimed approximately 655 000 lives worldwide in 2010. The Medicines for Malaria Venture suggests a "single-dose radical cure" as the ideal malaria treatment since rapid clearance of blood-stage parasites and symptom relief improves patient compliance and limits drug resistance. Thus, novel antimalarials should be rapid-acting and assessing their rate of activity is critical to drug discovery. Traditional evaluation of this rate by morphological assessments is flawed by highly subjective, operator-specific interpretations, mainly due to heterogeneous parasite morphology under routine culture conditions. This study aimed to develop an alternative, quantitative assay. Energy is vital for the growth and maintenance of all living organisms. Commercially available kits allow rapid quantification of the cell's energy currency, ATP. Therefore, quantification of parasite ATP shows potential for diagnosing abnormal parasite metabolism and the kinetics of drug action. In this study, a rapid protocol for detecting ATP in Plasmodium falciparum parasites using a luminescence-based kit was developed and optimised. Furthermore, luciferase-expressing transgenic parasites, in which luciferase activity is detected using a similar kit, were acquired. The utility of both methods for evaluating the rate of drug-induced stress was explored using antimalarials with varying modes of action and, presumably, rates of activity. Results showed that parasite ATP remained unchanged, increased or decreased during drug exposure. Morphological examinations by light microscopy and a Recovery assay, aided interpretation of the drug-induced changes in parasite ATP. These investigations suggested that unchanged parasite ATP levels reflect poor drug action, increased ATP levels indicate a stress response and partially compromised viability, while significantly reduced ATP reflects severely compromised viability. Concerning the Luciferase assay, parasite luciferase activity decreased during drug exposure, even in the presence of proteasome inhibitors. Changes in parasite ATP and luciferase activity occurred at rates which suggested that chloroquine is slow-acting, mefloquine has a moderate rate of activity and artemisinin is rapid-acting. These findings are compatible with the expected rates of activity of these established antimalarials. Hence, measurement of parasite ATP and/or luciferase activity may support assessments of parasite health and the kinetics of antimalarial action during drug discovery

Protein kinases are important regulatory enzymes in signal transduction and in cell regulation. Understanding inhibition mechanisms of kinases is important for the further development of new therapies for cancer and inflammatory diseases. I have developed a statistical approach based on the Mantel test to find the relationship between the shapes of ATP binding sites and their affinities for inhibitors. My shape-based dendrogram shows clustering of the kinases based on similarity in shape. I investigate the pocket in terms of conservation of surrounding amino acids and atoms in order to identify the key determinants of ligand binding. I find that the most conserved regions are the main chain atoms in the hinge region and I show that the tetrahydropyran ring of staurosporine causes induced-fit of the glycine rich loop. I apply multiple linear regression to select distances measured between the distinctive parts of residues which correlate with the binding constants. This method allows me to understand the importance of the size of the gatekeeper residue and the closure between the first glycine of the GXGXXG motif and the aspartate of the DFG loop, which act together to promote tight binding to staurosporine. I also find that the greater the number of hydrogen bonds made by the kinase around the methylamine group of staurosporine, the tighter the binding to staurosporine. The website I have developed allows a better understanding of cross reactivity and may be useful for narrowing down the options for a synthetic strategy to design kinase inhibitors.