The Regulation of Sleep and Circadian Rhythms: The Role of Melatonin and Adenosine in Zebrafish

Sleep is a highly conserved behavioral state whose regulation is still unclear. In this thesis I initially briefly introduce the known sleep circuitry and regulation in vertebrates, and why zebrafish is seen as a good model to study sleep-regulation. I describe the existing two-process model of sleep regulation, which posits that the two processes C (circadian) and S (homeostatic) control timing of sleep-wake behavior. I then study the role melatonin plays in the circadian regulation of sleep using zebrafish. Firstly, we find that the absence of melatonin results in a reduction of sleep at night, establishing that endogenous melatonin is required for sleep at night. Secondly, melatonin mutants show a reduction in sleep in animals with no functional behavioral rhythms suggesting that melatonin does not require intact circadian rhythms for its effect on sleep. Thirdly, melatonin mutants do not exhibit any changes in circadian rhythms, suggesting that the circadian clock does not require melatonin for its function. Fourthly, we find that in the absence of melatonin, there is no rhythmic expression of sleep, suggesting that melatonin is the output molecule of process C. Lastly, we describe a connection between adenosine signaling (output molecules of process S), and melatonin. Following this we proceed to study the role adenosine signaling plays in sleep-wake behavior. We find that firstly, adenosine receptor A1 and A2 are involved in sleep- wake behavior in zebrafish, based on agonist/antagonist behavioral results. Secondly, we find that several brain regions such as PACAP cells in the rostral midbrain, GABAergic cells in the forebrain and hindbrain, Dopamine and serotonin cells in the caudal hypothalamus and sox2 cells lining the hindbrain ventricle are activated in response to the A1 antagonist and VMAT positive cells are activated in response to the A2A agonist, suggesting these areas are involved in adenosine signaling in zebrafish. Thirdly, we find that knocking out the zebrafish adenosine receptors has no effect on sleep architecture. Lastly, we find that while the A1 agonist phenotype requires the zfAdora1a receptor, the antagonist and the A2A agonist behavioral phenotypes are not mediated by the zfAdora1a, zfAdora1b and zfAdoraA2Aa, zfAdora2Ab receptors respectively.

A tool for better management of water quality during periods of algal blooms: the case of the River Oise

Like other rivers in the Paris area, the Oise is subject to important seasonal algal blooms. This eutrophication generates notable problems for the production of drinking-water from a treatment plant on the river at Méry. A mathematical model has been developed to simulate variation in water quality in a pre-treatment storage basin, and another model is currently being adapted to model the River Oise. Integration of the two models should provide a comprehensive tool for predicting variations of phytoplankton and water-quality parameters associated with algal blooms. This will be a decision-aid for optimizing control of the treatment process for providing potable water.

Part I. Regulation of development in the cellular slime mold Dictyostelium discoideum. Part II. Polysomes and RNA synthesis during early development of the surf clam Spisula solidissima

Part I. The cellular slime mold Dictyostelium discoideum is a simple eukaryote which undergoes a multi-cellular developmental process. Single cell myxamoebae divide vegetatively in the presence of a food source. When the food is depleted or removed, the cells aggregate, forming a migrating pseudoplasmodium which differentiates into a fruiting body containing stalk and spore cells. I have shown that during the developmental cycle glycogen phosphorylase, aminopeptidase, and alanine transaminase are developmentally regulated, that is their specific activities increased at a specific time in the developmental cycle. Phosphorylase activity is undetectable in developing cells until mid-aggregation whereupon it increases and reaches a maximum at mid-culmination. Thereafter the enzyme disappears. Actinomycin D and cycloheximide studies as well as studies with morphologically aberrant and temporally deranged mutants indicate that prior RNA and concomitant protein synthesis are necessary for the rise and decrease in activity and support the view that the appearance of the enzyme is regulated at the transcriptional level. Aminopeptidase and alanine transaminase increase 3 fold starting at starvation and reach maximum activity at 18 and 5 hours respectively.

The cellular DNA s of D. discoideum were characterized by CsC1 buoyant density gradient centrifugation and by renaturation kinetics. Whole cell DNA exhibits three bands in CsCl: ρ = 1.676 g/cc (nuclear main band), 1.687 (nuclear satellite), and 1.682 (mitochondrial). Reassociation kinetics at a criterion of Tm -23°C indicates that the nuclear reiterated sequences make up 30% of the genome (Cot_1/2 (pure) 0.28) and the single-copy DNA 70% (Cot_1/2(pure) 70). The complexity of the nuclear genome is 30 x 10⁹ daltons and that of the mitochondrial DNA is 35-40 x 10⁶ daltons (Cot_1/2 0.15). rRNA cistrons constitute 2.2% of nuclear DNA and have a ρ = 1.682.

RNA extracted from 4 stages during developmental cycle of Dictyostelium was hybridized with purified single-copy nuclear DNA. The hybrids had properties indicative of single-copy DNA-RNA hybrids. These studies indicate that there are, during development, qualitative and quantitative changes in the portion of the single-copy of the genome transcribed. Overall, 56% of the genome is represented by transcripts between the amoeba and mid-culmination stages. Some 19% are sequences which are represented at all stages while 37% of the genome consists of stage specific sequences.

Part II. RNA and protein synthesis and polysome formation were studied during early development of the surf clam Spisula solidissima embryos. The oocyte has a small number of polysomes and a low but measurable rate of protein synthesis (leucine-³H incorporation). After fertilization, there is a continual increase in the percentage of ribosomes sedimenting in the polysome region. Newly synthesized RNA (uridine-5-³H incorporation) was found in polysomes as early as the 2-cell stage. During cleavage, the newly formed RNA is associated mainly with the light polysomes.

RNA extracted from polysomes labeled at the 4-cell stage is polydisperse, nonribosomal, and non-4 S. Actinomycin D causes a reduction of about 30% of the polysomes formed between fertilization and the 16-cell stage.

In the early cleavage stages the light polysomes are mostly affected by actinomycin.

Análisis del riesgo de contraparte a través del modelo estructural de Merton en el marco de Solvencia II

[ES] La entrada en vigor del proyecto de Solvencia II transformará por completo el sistema de determinación de las necesidades de capital del sector asegurador europeo. Recientemente se ha presentado el último estudio de impacto cuantitativo (QIS5), donde se establece la forma de cálculo del modelo estándar para la determinación de los requerimientos de capital. Este trabajo trata de analizar la adecuación de la calibración del riesgo de crédito de la contraparte mediante los modelos que se proponen en los últimos informes de impacto cuantitativo (cuarto y quinto). Para ello comparamos las necesidades de capital que se obtienen por ambas alternativas, frente a las que resultarían de aplicar un modelo de simulación basado en el enfoque estructural. Los resultados obtenidos muestran que el uso de probabilidades basadas en la metodología de Merton frente a aquellas basadas en ratings, dan lugar a requerimientos de capital sustancialmente mayores. Además, el modelo propuesto en QIS4 basado en la distribución de Vasicek no es adecuado cuando el número de contrapartes es reducido, situación habitual en el sector asegurador europeo. Por otra parte, la nueva propuesta (QIS5 o modelo de Ter Berg) es más versátil y adecuada que su antecesora pero requiere analizar con más detenimiento las hipótesis de calibración para de este modo aproximar mejor las estimaciones al riesgo realmente asumido.

Mass spectrometry coupled to imaging techniques: The better the view the greater the challenge

These are definitively exciting times for membrane lipid researchers. Once considered just as the cell membrane building blocks, the important role these lipids play is steadily being acknowledged. The improvement occurred in mass spectrometry techniques (MS) allows the establishment of the precise lipid composition of biological extracts. However, to fully understand the biological function of each individual lipid species, we need to know its spatial distribution and dynamics. In the past 10 years, the field has experienced a profound revolution thanks to the development of MS-based techniques allowing lipid imaging (MSI). Images reveal and verify what many lipid researchers had already shown by different means, but none as convincing as an image: each cell type presents a specific lipid composition, which is highly sensitive to its physiological and pathological state. While these techniques will help to place membrane lipids in the position they deserve, they also open the black box containing all the unknown regulatory mechanisms accounting for such tailored lipid composition. Thus, these results urges to different disciplines to redefine their paradigm of study by including the complexity revealed by the MSI techniques.

Regulação médica de urgências na região do Médio Paraíba: uma proposta para avaliação da implantação do serviço

O presente trabalho teve como objeto o desenvolvimento de uma proposta para avaliação da implantação da Central de Regulação Médica de Urgências (CRMU) vinculada ao Serviço de Atenção Móvel às Urgências da região do Médio Paraíba (SAMU 192 - MP). Para tal, propõe-se a criação de um modelo teóricológico que sirva como referência para elaboração de instrumentos e seleção de indicadores para avaliação da estrutura, processos e resultados da CRMU-MP, visando à melhor efetividade e eficiência desse serviço. O trabalho descreve as fases do processo de implantação e do funcionamento do SAMU-192 na região do Médio Paraíba. Aborda a sua importância para o sistema de saúde, visando minimizar quadros de demandas desnecessárias de urgência, com a diminuição dos fluxos informais de pacientes para grandes urgências hospitalares e préhospitalares da região, relatando os vários motivos relacionados à sobrecarga desses serviços na região: ineficiência da atenção básica; pronto-socorros préhospitalares e hospitalares de pequeno e médio porte sem retaguarda mínima de recursos diagnósticos e terapêuticos; fatores culturais; maior oferta das redes de alta complexidade; problemas relacionados à gestão. Questões sobre a estrutura da rede de urgências são mencionadas, evidenciando-se as deficiências do SUS: áreas físicas inadequadas e insuficientes, informalidade na contratação de recursos humanos além do despreparo dos profissionais que atuam nessa área. No cenário descrito, as ações de regulação despontam como ferramenta de defesa do cidadão, buscando garantir acesso ao meio mais adequado a suas necessidades. O desenho proposto (modelo teórico-lógico) descreve e identifica as etapas para avaliação do serviço (estrutura, processo e resultados), e também demonstra os diversos problemas encontrados na rede de atenção às urgências do Médio Paraíba. O trabalho faz uma revisão da literatura sobre os principais conceitos da regulação e da regulação médica de urgências; discorre sobre os aspectos da Política de Urgência e Emergência a partir de 2002, a inserção da região do Médio Paraíba e de sua rede de atenção às urgências neste contexto. Aborda os diversos conceitos da avaliação, com foco nos modelos teórico-lógicos e cita as estratégias metodológicas, empregando o modelo teórico-lógico como proposta para avaliar a implantação da CRMU. A última etapa trata da elaboração do modelo teórico-lógico, bem como de suas matrizes de avaliação e de seu elenco de indicadores.

Gain Scheduling Control of an Islanded Microgrid Voltage

The aim of this research study has been to design a gain scheduling (GS) digital controller in order to control the voltage of an islanded microgrid in the presence of fast varying loads (FVLs), and to compare it to a robust controller. The inverter which feeds the microgrid is connected to it through an inductance-capacitor-inductance (LCL) filter. The oscillatory and nonlinear behaviour of the plant is analyzed in the whole operating zone. Afterwards, the design of the controllers which contain two loops in cascade are described. The first loop concerns the current control, while the second is linked to the voltage regulation. Two controllers, one defined as Robust and another one as GS controller, are designed for the two loops, emphasizing in their robustness and their ability to damp the oscillatory plant behaviour. To finish, some simulations are carried out to study and compare the two kinds of controllers in different operating points. The results show that both controllers damp the oscillatory behaviour of the plant in closed loop (CL), and that the GS controller ensures a better rejection of current disturbances from FVLs.

European Market Infrastructure Regulation

El trabajo trata de dar unas nociones básicas de la regulación EMIR y de la descripción de la implantación de la solución que toma la empresa en la que trabajé en prácticas. Y lo he realizado en castellano

Passagens de idade: uma análise antropológica sobre as articulações entre o saber biomédico e o saber leigo no discurso de mulheres de camadas médias

Este trabalho se propõe ao estudo das articulações entre o saber biomédico e o saber leigo no discurso de mulheres de camadas médias urbanas da cidade do Rio de Janeiro, com idades compreendidas entre 40 e 60 anos. Toma como referência a produção sócioantropológica sobre gênero, corpo, menopausa, envelhecimento, bem como a que analisa a construção do fato científico. Atualmente é notável a crescente preeminência do discurso da biologia na determinação daquilo que seria da ordem do corpo feminino ou do masculino, o que certamente é perpassado por relações de gênero. A partir de uma perspectiva construcionista investiga o destaque conferido ao papel dos hormônios pela biomedicina, não apenas no funcionamento e na regulação do organismo da mulher, mas também no que diz respeito à forma pela qual ela irá vivenciar seu cotidiano. Além disso, busca apontar pistas na direção de problematizar a pregnância de certos dualismos como o que opõe o biológico ao social. Dualismos que revelam uma forma de organização binária hierárquica de pensamento, característica de nossa sociedade ocidental moderna. A noção de rede é um balizador importante para a compreensão mais ampla do pano de fundo do que informa as leituras vigentes acerca das ocorrências fisiológicas de mulheres e homens. Embora o campo investigado se apresente fluido e matizado, a intensidade do discurso hormonal biomédico se faz notar de modo potente nas narrativas, no que diz respeito aos corpos e às vidas das mulheres entrevistadas.