Preparação de nanoestruturas multifuncionais de polímero@ouro e preparação de filmes finos em multicamadas para biodeteção

Mestrado em Química Inorgânica e MateriaisO objetivo desta tese de mestrado era desenvolver nanoestruturas robustas e multifuncionais do tipo coroa@núcleo para servir como base num sensor biológico. A estabilidade e as propriedades óticas das nanopartículas de ouro (Au NPs), nomeadamente a ressonância de plasmão de superfície localizada (LSPR), tornam as excelentes candidatos para o desenvolvimento de biossensores baseados em SPR. No seguimento de um trabalho anterior, procedeu-se primeiro à otimização da encapsulação e funcionalização dasAu NPs. Em segundo lugar, preparou-se filmes finos de multicamadas que incorporam as nanopartículas encapsuladas. Au NPs (15 4 nm) foram sintetizadas pelo método de Turkevich, e sua superfície foi revestida por polimerização de transferência de cadeia reversível por adição-fragmentação (RAFT) assistida em encapsulante emulsão (REEP). Foram explorados dois tipos de nanoestruturas: i) nanoestruturas não funcionalizadas foram preparadas com sucesso adsorvendo um agente macroRAFT (MR) P(PEGA40)-TTC) não funcionalizado nas Au NPs, seguido pelo crescimento do bloco hidrofóbico usando uma mixtura (10:1 w/w) de metacrilato de metilo (MMA) e de acrilato de butilo (BA) a partir de MR@Au NPs; e ii) nanoestruturas funcionalizadas foram preparadas usando um agente MR funcionalizado com um bioreceptor (biotina) e um fluoróforo (isotiocianato de fluoresceína - FITC). Foram realizadas tentativas para crescer o bloco hidrofóbico apartir das nanoestruturas multifuncionais MR@Au com o objetivo de melhorar a sua resposta óptica aumentando a distância entre o núcleo e o fluoróforo. No entanto, concluiu-se que a estratégia seguida para preparar estas nanoestruturas multifuncionais tem de ser revista. O tamanho e a estabilidade das nanoestruturas de ouro foi avaliado por espectroscopia UV-Visível e fluorescência, STEM (microscopia eletrónica de transmissão de varrimento), DLS (dispersão dinâmica de luz) e medidas de potencial zeta. A caracterização química e estrutural dos polímeros foi feita por espectroscopias de 1H-RMN (ressonância magnética nuclear de protões) e de infravermelho, e cromatografia de permeação de gel – cromatografia de exclusão por tamanho (GPC-SEC). Em seguida, foram realizados testes de biodeteção usando a nanoestrutura multifuncional de coroa@núcleo no sentido de avaliar a sua resposta óptica na presença de um bioanalito específico - avidin. No entanto, estes testes não foram conclusivos devido às limitações da estratégia seguida para preparar as nanoestruturas multifuncionais. Paralelamente ao trabalho descrito acima, estudou-se a preparação de filmes finos usando o método camada a camada (Layer-by-Layber, LbL) baseado em interações electrostáticas, para isso foi usado o hidrocloreto de polialilamina (PAH) e as nanoestruturas de Au. Foram estudados alguns parâmetros nomeadamente o peso molecular, tempo de deposição, pH e força iónica, e o número de camadas. O objetivo final era depositar na última camada as nanoestruturas de ouro funcionalizadas com biotina-FITC. O procedimento para a deposição por LbL das NPs de Au encapsuladas foi realizado com sucesso, mas uma vez que os testes de biodeteção não foram conclusivos este último passo não foi efectuado.The aim of this master thesis was to develop robust, easily prepared multifunctional shell@core gold nanostructures to serve as a basis for customization into a specific biological sensor. The stability and optical properties of gold nanoparticles (Au NPs), namely the localized surface plasmon resonance (LSPR), makes them excellent candidates for the development of SPR-based biosensors. In this work, the preparation and functionalization of the gold nanoparticles was first optimized, on the basis of the previous work. Next, multilayered thin-films embedding these gold nanostructures were prepared. Au NPs (15 4 nm) were synthetized by the Turkevich method, and their surface was coated via reversible addition fragmentation chain transfer (RAFT) assisted encapsulating emulsion polymerization (REEP). The preparation of two types of nanostructures was explored: i) non-functionalized nanostructures were successfully prepared using the macroRAFT (MR) agent P(PEGA40)-TTC) adsorbed onto Au NPs, followed the growth of a hydrophobic block using a 10:1 (w/w) mixture of methyl methacrylate and butyl acrylate from MR@Au NPs; and ii) functionalized nanostructures were prepared using a MR agent functionalized with a bio-receptor (biotin) and a fluorophore (fluorescein isothiocyanate - FITC). Attempts to grow a hydrophobic block from the multifunctional MR@Au NPs were carried out to enhance the optical response by increasing the distance between the core and fluorophore. Yet, the synthetic strategy followed to prepare this type of multifunctional nanostructures needs to be revised. The size and stability of gold nanostructures were characterized by UV-Visible and fluorescence spectroscopy, STEM (scanning transmission electron microscopy), DLS (dynamic light scattering) and Zeta potential measurements. The chemical and structural characterization of polymers was carried out by 1HNMR (proton nuclear magnetic resonance), infrared spectroscopies and gel permeation chromatography – size exclusion chromatography (GPC-SEC). Then, biosensing tests were performed on the multifunctional shell@core nanostructures synthetized to assess their optical response towards a specific bioanalyte - avidin. However, these tests were inconclusive due to the limitations of the synthetic strategy followed to prepare the Au nanostructures. Parallel to the synthetic work described above, the preparation of thin films via electrostatic interactions using the Layer-by-Layer (LbL) method was studied, using poly(allylamine hydrochloride) (PAH) and carboxylic acid terminated non-functionalized Au nanostructures. Various parameters were studied such as molecular weight, deposition time, pH and ionic strength, as well as the number of layers. The final goal was to deposit the biotin-FITC-functionalized gold nanostructure as last layer. The optimized procedure for LbL deposition of the encapsulated Au NPs was fully accomplished, in view of the fact that the biosensing tests were inconclusive, this last step was not carried out

Sudden Death of Cardiac Origin and Psychotropic Drugs

Mortality rate is high in psychiatric patients versus general population. An important cause of this increased mortality is sudden cardiac death (SCD) as a major side-effect of psychotropic drugs. These SCDs generally result from arrhythmias occurring when the posology is high and may attain a toxic threshold but also at dosages within therapeutic range, in the presence of risk factors. There are three kinds of risk factors: physiological (e.g., low cardiac rate of sportsmen), physiopathological (e.g., hepatic insufficiency, hypothyroidism) and “therapeutic” (due to interactions between psychotropic drugs and other medicines). Association of pharmacological agents may increase the likelihood of SCDs either by (i) a pharmacokinetic mechanism (e.g., increased torsadogenic potential of a psychotropic drug when its destruction and/or elimination are compromised) or (ii) a pharmacodynamical mechanism (e.g., mutual potentiation of proarrhythmic properties of two drugs). In addition, some psychotropic drugs may induce sudden death in cases of pre-existing congenital cardiopathies such as (i) congenital long QT syndrome, predisposing to torsade de pointes that eventually cause syncope and sudden death. (ii) A Brugada syndrome, that may directly cause ventricular fibrillation due to reduced sodium current through Nav1.5 channels. Moreover, psychotropic drugs may be a direct cause of cardiac lesions also leading to SCD. This is the case, for example, of phenothiazines responsible for ischemic coronaropathies and of clozapine that is involved in the occurrence of myocarditis. The aims of this work are to delineate: (i) the risk of SCD related to the use of psychotropic drugs; (ii) mechanisms involved in the occurrence of such SCD; (iii) preventive actions of psychotropic drugs side effects, on the basis of the knowledge of patient-specific risk factors, documented from clinical history, ionic balance, and ECG investigation by the psychiatrist

‘MUTIRÃO AGROFLORESTAL’: HERRAMIENTA DE RED DE AGROFORESTERÍA DEL VALE DO PARAÍBA, BRASIL

Difundir los sistemas agroforestales (SAFs) en la cuenca del río Paraíba do Sul, Estado de São Paulo, Brasil, es el propósito de esfuerzo conjunto en Polo Regional/APTA, en Pindamonhangaba. Los SAFs se basan en consorcios de cultivos agrícolas, arbustos y árboles, aprovechando los mismos recursos (agua, luz y nutrientes). Entre los años 2010-2013, unas 330 personas de diversos orígenes participaron en esfuerzos conjuntos agroforestales. La metodología participativa incluye la planificación para la preparación y manejo de los SAFs, después de conciencia a través de conferencias y excursión. El método "mutirão agroforestal” promueve el intercambio de conocimiento, el rescate de los saberes populares y los recursos genéticos. El enfoque de SAFs en la restauración de las áreas ribereñas con producción diversificada amplificando la biodiversidad de la selva atlántica. La producción se basa en especies comestibles, entre ellos: Maranta arundinaceae, Colocasia esculenta, Pereskia sp., Talinum paniculatum, Xanthosoma sagittifolium, Manihot esculenta, plátano resistente - Musa sp.; de árboles nativo para madera (Calophyllum brasiliense e Centrolobium tomentosum), frutas (Euterpe edulis, Rolinia mucosa) y plantas fertilizantes (Cajanus cajan, Flemingia macrophylla, Tithonia diversifolia, Gliricidia sepium, Sesbania sp., Inga sp.). Se evalúan indicadores de sostenibilidad para balisar la gestión: resistencia a la penetración de una barra de hierro, tasa de cobertura del suelo por la proyección de la plantas de dosel, cantidad y calidad de vegetación espontánea, la presencia de organismos vivos en la capa superior del suelo, contenido de materia orgánica por la reacción del suelo con peróxido de hidrógeno, calidad de especies de anclaje y la cantidad de hojarasca del suelo. Como resultado, un grupo multidisciplinario ha sido articular la formación de Red de Agroforestería del Vale do Paraíba, de difundir los SAFs en la cuenca y los recursos genéticos de estas unidades pasan a los productores

‘Mutirão Agroflorestal’: herramienta de red de agroforestería del Vale do Paraíba, Brasil

Difundir los sistemas agroforestales (SAFs) en la cuenca del río Paraíba do Sul, Estado de São Paulo, Brasil, es el propósito de esfuerzo conjunto en Polo Regional/APTA, en Pindamonhangaba. Los SAFs se basan en consorcios de cultivos agrícolas, arbustos y árboles, aprovechando los mismos recursos (agua, luz y nutrientes). Entre los años 2010-2013, unas 330 personas de diversos orígenes participaron en esfuerzos conjuntos agroforestales. La metodología participativa incluye la planificación para la preparación y manejo de los SAFs, después de conciencia a través de conferencias y excursión. El método "mutirão agroforestal” promueve el intercambio de conocimiento, el rescate de los saberes populares y los recursos genéticos. El enfoque de SAFs en la restauración de las áreas ribereñas con producción diversificada amplificando la biodiversidad de la selva atlántica. La producción se basa en especies comestibles, entre ellos: Maranta arundinaceae, Colocasia esculenta, Pereskia sp., Talinum paniculatum, Xanthosoma sagittifolium, Manihot esculenta, plátano resistente - Musa sp.; de árboles nativo para madera (Calophyllum brasiliense e Centrolobium tomentosum), frutas (Euterpe edulis, Rolinia mucosa) y plantas fertilizantes (Cajanus cajan, Flemingia macrophylla, Tithonia diversifolia, Gliricidia sepium, Sesbania sp., Inga sp.). Se evalúan indicadores de sostenibilidad para balisar la gestión: resistencia a la penetración de una barra de hierro, tasa de cobertura del suelo por la proyección de la plantas de dosel, cantidad y calidad de vegetación espontánea, la presencia de organismos vivos en la capa superior del suelo, contenido de materia orgánica por la reacción del suelo con peróxido de hidrógeno, calidad de especies de anclaje y la cantidad de hojarasca del suelo. Como resultado, un grupo multidisciplinario ha sido articular la formación de Red de Agroforestería del Vale do Paraíba, de difundir los SAFs en la cuenca y los recursos genéticos de estas unidades pasan a los productores

Genetic associations of protein-coding variants in human disease.

Genome-wide association studies (GWAS) have identified thousands of genetic variants linked to the risk of human disease. However, GWAS have so far remained largely underpowered in relation to identifying associations in the rare and low-frequency allelic spectrum and have lacked the resolution to trace causal mechanisms to underlying genes1. Here we combined whole-exome sequencing in 392,814 UK Biobank participants with imputed genotypes from 260,405 FinnGen participants (653,219 total individuals) to conduct association meta-analyses for 744 disease endpoints across the protein-coding allelic frequency spectrum, bridging the gap between common and rare variant studies. We identified 975 associations, with more than one-third being previously unreported. We demonstrate population-level relevance for mutations previously ascribed to causing single-gene disorders, map GWAS associations to likely causal genes, explain disease mechanisms, and systematically relate disease associations to levels of 117 biomarkers and clinical-stage drug targets. Combining sequencing and genotyping in two population biobanks enabled us to benefit from increased power to detect and explain disease associations, validate findings through replication and propose medical actionability for rare genetic variants. Our study provides a compendium of protein-coding variant associations for future insights into disease biology and drug discovery

Re-examining the transition into the N=20 island of inversion: structure of 30Mg

Intermediate energy single-neutron removal from $^{31}$Mg has been employed to investigate the transition into the N=20 island of inversion. Levels up to 5~MeV excitation energy in $^{30}$Mg were populated and spin-parity assignments were inferred from the corresponding longitudinal momentum distributions and $\gamma$-ray decay scheme. Comparison with eikonal-model calculations also permitted spectroscopic factors to be deduced. Surprisingly, the 0$^{+}_{2}$ level in $^{30}$Mg was found to have a strength much weaker than expected in the conventional picture of a predominantly $2p - 2h$ intruder configuration having a large overlap with the deformed $^{31}$Mg ground state. In addition, negative parity levels were identified for the first time in $^{30}$Mg, one of which is located at low excitation energy. The results are discussed in the light of shell-model calculations employing two newly developed approaches with markedly different descriptions of the structure of $^{30}$Mg. It is concluded that the cross-shell effects in the region of the island of inversion at Z=12 are considerably more complex than previously thought and that $np - nh$ configurations play a major role in the structure of $^{30}$Mg.Comment: Physics Letters B, Volume 779, 10 April 2018, Pages 124-12

Effects of low [K+]o on the electrical activity of human cardiac ventricular and Purkinje cells: Human ventricular and Purkinje cells in low [K+]o

International audienceVentricular and Purkinje action potentials were recorded with a microelectrode in a strip of human papillary muscle. Lowering the K-content of the superfusing solution from 5.9 to 0.5 mmol.litre-1 at 37 degrees C hyperpolarised ventricular diastolic potential steadily as long as [K+]o was low (up to 70 min tested). Ventricular action potentials were transiently lengthened and then shortened. A positive inotropic effect was noted and attributed to Na-K pump inhibition since it was reversed by the addition of 2 mmol.litre-1 thallous chloride to the low [K+]o solution. Beyond 40 min, transient depolarisations and after-contractions were found. During the first minutes in low [K+]o, Purkinje diastolic potential was hyperpolarised and the action potential was lengthened at all levels of repolarisation. Afterwards, the Purkinje diastolic potential suddenly depolarised by 30 mV. Restoration of the control solution caused a slow repolarisation and then a sudden return of the diastolic potential to near control value. This was reproduced during drive (38 stim.min-1) and at rest. At the depolarised level of potential, stimulation elicited slow action potentials with diastolic slow depolarisation and spontaneous oscillations of potential appeared at rest. In Purkinje cells, increasing concentrations of tetrodotoxin from 10(-7) to 8 X 10(-6) mol.litre-1 in the control solution shifted the diastolic potential in negative direction by a few mV and shortened the action potential duration at all levels of repolarisation. The possible implications of these phenomena in the genesis of some cardiac arrhythmias are discussed

Localization of K(+) channels in the tubules of cardiomyocytes as suggested by the parallel decay of membrane capacitance, IK(1) and IK(ATP) during culture and by delayed IK(1) response to barium.: Inward Rectifier Currents and Cardiac T-tubules

International audienceAdult ventricular myocytes lose T-tubules over few days in culture, which causes the loss of about 60% of the cell membrane capacitance (Cm) (Mitcheson et al., 1996). In this study, we have measured, in whole-cell voltage-clamped rabbit right ventricular myocytes at 0, 1, 2 and 3-5 days of culture (nine to 20 myocytes at each age) in a defined Dulbecco's modified Eagle's medium, the value of Cm and the magnitudes of the background inward rectifier current (IK(1)) and of the 2,4-dinitrophenol-induced ATP-sensitive potassium current (IK(ATP)). Cm, IK(1) and IK(ATP) all had decreased significantly by 51, 83 and 88%, respectively after 4 days of culture. Analysis using a single exponential decay function of time gave time constants of, 2.6+/-0.2, 2.2+/-0.5 and 2.4+/-0.4 days, respectively. Linear regressions of IK(1) and IK(ATP) versus Cm had regression coefficients of 0.93 and 0. 98, respectively. These observations are consistent with a strong link of the decay of IK(1) and IK(ATP) currents to that of Cm. Furthermore, the time course of changes in IK(1) when an external blocker (100 microm BaCl(2)) was applied and washed by local perfusion (95% change in 50 ms) agrees with a model including a diffusion time constant of 300 ms. This value is consistent with the known kinetics of diffusion of divalent cations in the T-tubules. Taken together, these results could be explained by the localization of a major part of the IK(1) and IK(ATP) currents of ventricular cardiomyocytes in the T-tubules. As a consequence, transient accumulation of K(+) ions in cardiac T-tubules may take place and modulate excitation-contraction coupling