Molecular Modelling of a novel G-quadruplex structure and its interaction with ligands

DNA can exist under many different forms. Lately, G-quadruplexes, which are one example of the non-canonical DNA forms, have been getting a lot of attention due to the role they play in certain biological processes and as potential targets for therapeutic interventions. For example, these structures can exist in certain parts of the telomeres, structures responsible for cell replication. In cancer cells, if the enzyme telomerase could be inhibited, by inducing the formation of a G-quadruplex structure in guanine-rich telomere sequences, the spread of cancer cells would cease. For this and other reasons, it becomes important to be able to induce the formation of G-quadruplex structures and/or stabilize them, and one of the ways of doing so consists of targeting these sequences with ligands that have good affinity to G-quadruplex structures. However, few G-quadruplex ligands demonstrated the needed properties to fulfill the clinical needs, and further efforts to determine which would be better suited to target any particular sequence are needed. This work aimed at comparing the affinity to the pre-miR-149 G-quadruplex structure of seven promising ligands found in the literature, through the latest techniques fit for that purpose. The seven ligands tested were: [16]phenN2, [32]phen2N4, phen-DC3, pyridostatin, acridine orange derivatives C8 and C8-NH2 and L-arginine. Firstly, they underwent computational tests, with the molecular structure of the quadruplex and the ligand being simulated, and their optimal binding site and conformation found. Their binding energies were compared, and they underwent molecular dynamics runs to simulate their behavior in an environment with solvent, followed by another binding energy comparison. The trend obtained in order of decreasing binding affinity was: pyridostatin > [32]phen2N4 > [16]phenN2 > Phen-DC3 > L-arginine > C8 > C8-NH2. Biophysical techniques were then performed, to determine the binding affinities experimentally. First, circular dichroism spectroscopy and melting studies (performed on four ligands) established the following trend: C8 > pyridostatin > C8-NH2 > [16]phenN2. Fluorescence spectroscopy titration (performed on three) revealed a similar trend: C8 > C8-NH2 > [16]phenN2. Lastly, affinity chromatography experiments were held to test how other DNA sequences would bind to C8-NH2. The results revealed that the ligand has better binding affinity with parallel quadruplexes over antiparallel ones, and poor binding with a duplex sequence. Overall, the best ligands identified for binding to the G-quadruplex structure were the acridine orange derivatives C8 and C8-NH2, and pyridostatin. These three ligands should be considered prime candidates for further research in this area.ADN pode existir sob a forma de diversas estruturas, contrariamente ao que a vasta maioria da população pensa, ao imaginar a dupla hélice de Watson e Crick. Uma das formas que tem sido mais investigada ultimamente consiste no G-quadruplex. Esta estrutura não canónica do DNA ocorre quando guaninas se emparelham e organizam em estruturas cíclicas através de pontes de hidrogénio Hoogsteen, chamadas G-quartetos. Estas estruturas formam-se por empilhamento p-p entre elas próprias, originando o G-quadruplex, desde que haja um catião (preferivelmente K+) para assumir uma localização central entre todos os quartetos. Estas estruturas desempenham funções importantes a nível de regulação da transcrição e replicação do DNA. Alguns estudos indicam também que podem ser relevantes a nível de manutenção do DNA, e que várias secções do DNA humano se encontram num estado de equilíbrio entre a forma de G-quadruplex e duplex. São também considerados alvos para certas abordagens terapêuticas a nível do cancro. Por exemplo, vários oncogenes como c-kit e c-myc têm a capacidade de formar G-quadruplexes nos seus promotores. Controlando a forma que estes genes assumem, seria possível controlar a sua transcrição, e possivelmente impedir a formação de cancro. Outra possibilidade cinge-se à inibição da telomerase, uma enzima responsável pela replicação celular, que está sobreexpressa em células cancerígenas. Se uma parte do telómero assumir uma estrutura em G-quadruplex, a ação desta enzima fica inibida, efetivamente parando a progressão do cancro. Portanto, torna-se necessário induzir e estabilizar a formação de estruturas do G-quadruplex. A estratégia é utilizar ligandos que interajam por interações intermoleculares de forma a estabilizar a estrutura do G-quadruplex, e outra topologia que esteja em equilíbrio. No entanto, analisando a literatura, conclui-se que apenas alguns grupos de ligandos são efetivamente ligandos de G-quadruplex. Este trabalho de investigação teve como objetivo comparar 7 ligandos promissores da estrutura de G-quadruplex designada por pre-miR-149 literatura. Os ligandos selecionados foram macrociclos derivados de fenantrolina ([16]phenN2, [32]phen2N4, Phen-DC3, e derivados de laranja de acridina C8 e C8-NH2. Determinou-se a afinidade e a estabilização destes ligandos com a estrutura do RNA G-quadruplex, a pre-miR-149. Isso será feito em duas etapas principais. Primeiro, foram realizadas simulações computacionais para determinar quais os ligandos mais promissores e quais os seus métodos de interação com a estrutura G-quadruplex. Estas dividiram-se em três passos: primeiro, foram geradas as estruturas da sequência e de cada ligando em software adequado. Segundo, foram feitas simulações de docking de modo a averiguar os locais de ligação de cada ligando ao G-quadruplex, e a conformação e interações entre o ligando e o quadruplex, sendo também calculadas energias de ligação entre o ligando e o G-quadruplex. Finalmente, foram feitas simulações de dinâmica molecular sobre como essa conformação evoluiria num ambiente fisiológico simulado e calculadas novas energias de ligação, que comparadas entre si, revelam diferenças de afinidades entre os ligandos. Após estas técnicas computacionais, foram executadas técnicas biofísicas, como espetroscopia de dicroísmo circular e estudos de desnaturação térmica, e espectroscopia de fluorescência para determinar experimentalmente as afinidades de cada ligando para com a estrutura escolhida. Foram também executadas experiências de cromatografia de afinidade para determinar o comportamento de um ligando para com sequência do RNA G-quadruplex, a pre-miR-149. O programa usado para avaliar as conformações iniciais gerou estruturas demasiado rígidas e pouco flexíveis com os ligandos macrocíclicos [16]phenN2 e [32]phen2N4. As energias de ligação obtidas revelaram a nível de afinidade a seguinte ordem decrescente: piridostatina > [32]phen2N4 > [16]phenN2 > PhenDC3 > L-arginina > C8 > C8-NH2. Esta tendência não foi a mesma verificada experimentalmente, e logo, foi descartada. A nível destas experiências, retiram-se maioritariamente apenas as conformações dos ligandos que não são macrociclos. A nível das experiências de dicroísmo circular mencionadas, as variações de temperatura de desnaturação térmica ligando-quadruplex foram diferentes,verificando-se a seguinte ordem: C8 > piridostatina > C8-NH2 > [16]phenN2. Seguidamente, foram realizadas titulações por espectroscopia de fluorescência as quais revelaram a seguinte tendência: C8 > C8-NH2 > [16]phenN2. De notar que apenas quatro dos sete ligandos ([16]phenN2, [32]phen2N4, C8 and C8-NH2) possuíam fluorescência intrínseca, e que desses, apenas estes três puderam ser selecionados. Estes resultados mostraram que a piridostatina, e derivados de laranja de acridina C8 e C8-NH2 apresentaram maior afinidade para esta estrutura de G-quadruplex. Por último, os resultados de cromatografia de afinidade revelaram que o ligando C8-NH2 tem maior afinidade com o RNA G-quadruplex pre-miR-149 . Das seis sequências testadas, três delas (c-myc, c-kit e pre-miR-149) formam G-quadruplexes com topologia paralela, e tiveram tempos de retenção mais altos. Outras sequências (TBA e AG23) formam G-quadruplexes com topologia antiparalela, e mostram tempos de retenção mais baixos. A sequência ds26 (duplex) teve o tempo de retenção mais baixo. Conclui-se que este ligando tem maior especificidade para com G-quadruplexes com topologia paralela em detrimento do duplex. As simulações de docking corroboram esta conclusão. Deste modo, conclui-se que os melhores ligandos a nível de afinidade para com a sequência pre-miR-149 são os derivados de laranja de acridina C8 e C8-NH2 e a piridostatina, de modo que futura investigação nesta área deve considerar estes três como fortes candidatos a ligandos de RNA G-quadruplex

Bolsa de objectos digitais

O presente trabalho propõe a concepção e implementação de um sistema de recuperação de informação de diversos Objectos Digitais (objecto originalmente criado no meio informático, como por exemplo um documento, um vídeo…) com recurso a anotações/metadados descritivos do objecto (dados que descrevem outros dados). Para fomentar o processo de preenchimento dos metadados é proposto um mecanismo de créditos para estimular o preenchimento cooperativo entre utilizadores e autores de Objectos Digitais (OD), com base na metáfora da bolsa de valores. Deste processo de anotações resulta incerteza, ou seja, o mesmo objecto pode ser descrito de forma diferente por utilizadores distintos. Este sistema propõe uma forma de lidar com esta incerteza, recorrendo ao conceito de base de dados probabilística que não é mais que associar a cada termo armazenado, um valor (peso do termo) entre 0 e 1, a que demos o nome de peso do termo. Deste modo, foi também proposto um modelo de cálculo do peso dos termos descritivos dos OD baseado no feedback fornecido pelos utilizadores. Recorrendo ao peso dos termos, foi ainda possível determinar a relevância de um OD quando confrontado com uma pergunta dando assim a possibilidade a um utilizador, recuperar/pesquisar OD de qualquer tipo, ordenados por um valor de relevância. Para além da concepção e correspondente implementação do BOD, este trabalho discute ainda diferentes cenários de aplicação do sistema, ilustrando consequentemente a sua versatilidade e flexibilidad

Criteria Assessment of Energy Carrier Systems Sustainability

The sustainability assessment of an energy conversion process into electrical energy is carried out in technological, economical, environmental and social dimensions. A solid basis for a state-of-the-art interdisciplinary assessment using data obtained from the literature supports the sustainability comparison. Thus, indicators that best describe the technologies and that are related to each of the abovementioned dimensions are defined to quantify the sustainability of energy carrier systems. These indicators are: efficiency of electricity generation, lifetime, energy payback time, capital cost, electricity generation cost, greenhouse gases emissions during full life cycle of the technology, land requirements, job creation and social acceptance.info:eu-repo/semantics/publishedVersio

Seismic Response Assessment of Underground Structure Cross-Sections Using Response Spectra

A modelling technique of the cross-sections of underground structures based on the combination of modal shapes by response spectra is developed and calibrated according to the vertically propagating shear-wave model. Using the developed technique, the influence of several parameters in the flexibility index (ground-structure relative stiffness) were studied. To point out a few practical implications and simplifications introduced in the model, essentially related to the material behaviour adopted, four cases were studied in detail. To cover the lack of compatibility between the damping of the response spectrum, the ground stiffness and the strain level, more analyses with compatible properties were conducted

Three-Dimensional CFD modelling and analysis of the thermal entrainment in open refrigerated display cabinets

This study presents a three-dimensional Computational Fluid Dynamics (CFD) simulation of the air flow pattern and the temperature distribution in a refrigerated display cabinet. The thermal entrainment is evaluated by the variations of the mass flow rate and thermal power along and across the air curtain considering the numerical predictions of abovementioned properties. The evaluation on the ambient air velocity for the three-dimensional (3D) effects in the pattern of this type of turbulent air flow is obtained. Additionally, it is verified that the longitudinal air flow oscillations and the length extremity effects have a considerable influence in the overall thermal performance of the equipment. The non uniform distribution of the air temperature and velocity throughout the re-circulated air curtain determine the temperature differences in the linear display space and inside the food products, affecting the refrigeration power of display cabinets. The numerical predictions have been validated by comparison with experimental tests performed in accordance with the climatic class n.º 3 of EN 441 Standard (Tamb = 25 ºC,φamb = 60%; vamb = 0,2 m s-1). These tests were conducted using the point measuring technique for the air temperature, air relative humidity and air velocity throughout the air curtain, the display area of conservation of food products and nearby the inlets/outlets of the air mass flow.info:eu-repo/semantics/publishedVersio

Experimental analysis of the thermal entrainment three dimensional effects in re-circulated air curtains

The application of air curtains devices to HVAC&R systems have been developed in order to improve the thermal and mass separation between two contiguous spaces with differentiated air behaviours. The refrigeration equipments installed inside commercial spaces and open to the surroundings suffer alterations of their thermal behaviours and energy efficiency due to variations in magnitude and orientation of the air velocity. The location of the air conditioning system discharge grilles, the mass flows rates originated by pressure differences due to openings to the external environment and the air flow perturbation due to the circulation of the consumers nearby the opening of the display cases, among others, affect the re-circulated air curtain behaviour and the overall performance of the equipment.This study presents the experimental results obtained from tests carried out for the climatic class n.º 3 of EN 441 (Tamb = 25 ºC, φamb = 60%; vamb = 0,2 m/s) and the analysis of the threedimensional effects observed in the thermal interaction through the air curtains installed in open vertical refrigerated display cases. The obtained results are related to the air temperature, air relative humidity and air velocity measurements fields throughout the air curtain. From the analysis of the obtained results some comparable effects in the fields of the same properties for the display space of the exposition and conservation of food products and nearby the inlets/outlets of the air mass flow, can be sorted out. The evaluation of the ambient air velocity three-dimensional effects in the pattern of this type of turbulent air flow is obtained. Additionally, it is verified that the longitudinal airflow fluctuations and the length extremity effects of the display case have a considerable influence in the overall thermal performance of the equipment. The non-uniform distribution of the air temperature, relative humidity and velocity throughout the air curtain determines temperature differences in the linear dimension display space and inside the food products, affecting the correct behaviour of the display cases. Besides that, this study helps to show some of the fragilities of the numerical models bi-dimensional formulation.info:eu-repo/semantics/publishedVersio

Sustainability quantification of renewable energy conversion systems for electricity supply

The sustainability of renewable energy conversion systems for electricity supply is quantified and compared. The energy conversion systems considered are: wind, photovoltaic, geothermal, hydro, hydrogen, ocean (wave and tidal power) and nuclear. The sustainability analysis is performed covering technological, economic, environmental and social aspects. The indicators selected to characterize and quantify the sustainability of each subgroup are: efficiency, lifetime, electricity generation cost, capital cost, CO2 emissions, area occupied, employment creation and social acceptance. Membership functions are applied to determine the sustainability index that quantifies how sustainable is each energy conversion system depending on the most relevant indicator. This procedure includes a weighting coefficient that varies in each case study to magnify the importance of one indicator relatively to the others. Sustainability indices are compared in order to assess the energy conversion systems mix for electricity supply more sustainable now and in the near future. Comparing the several case studies, the global sustainability indicators suggest that the mix of energy conversion systems for electricity supply should be based on hydro, wind and nuclear systems.info:eu-repo/semantics/publishedVersio

Contribution for a better understanding of the technological sustainability in electrical energy production through photovoltaic cells

The conversion of solar energy into electricity reveals a huge importance in the production of “clean” energy , mainly when applied to decentralized production systems (micro-generation). However, there is the need to develop and optimize these processes in order to turn it more sustainable in economic and technological scoops. The main purpose of this work is to study the solar energy conversion into electricity through photovoltaic cells, characterizing the process efficiencies. This study intends to evaluate the energetic and exergetic efficiencies defining them as indicators in the formulation of a sustainability index. All the procedures are in a theoretical scope with an illustrative example in the end of this work.info:eu-repo/semantics/publishedVersio

Uma causa muito rara de espasmos infantis

Psychomotor development regression or delay associated with epilepsy represent a diagnostic challenge. The diagnostic approach should take into account age group, epileptic syndrome, physical and neurological data, and organ and/or system involvement. Herein is reported the case of a toddler for whom hair development, epileptic seizure evolution, and electroencephalographic findings were key for Menkes kinky hair disease diagnosis. The typical electroclinical evolution in this syndrome has rarely been previously reported. A 22-month-old boy, born at 35 weeks, was admitted to the hospital by the age of two months due to epileptic seizures. Physical examination revealed dysmorphic facial features, pectus excavatum, and inguinal hernias. Antiepileptic drugs were initiated and one month later the patient was readmitted with recurrent epileptic seizures. Transfer to a hospital with Pediatric Neurology support was required, where light-toned and pleated skin, sparse hair, failure to thrive, and axial hypotonia were remarked. Initial investigation with general metabolic, neuroimaging, ophthalmological, and microarray study revealed no changes. Electroencephalograms were markedly abnormal, initially with focal changes and later with hypsarrhythmia. Considering the patient’s phenotype, copper serum level was analysed, with null value. Molecular study confirmed Menkes kinky hair disease and copper histidine therapy was initiated. Menkes kinky hair disease should be considered in infants with global developmental delay, severe hypotonia, refractory epilepsy, and typical hair and skin changes occurring early in life. However, neonatal diagnosis is hampered by age-unspecific signs and symptoms. Despite being a rare and fatal entity, timely diagnosis allowing early therapy institution and avoiding unnecessary additional tests and prompt genetic counseling are of utmost importance.info:eu-repo/semantics/publishedVersio

Testing four methods to assess leaf area in young olive trees

Assessment of leaf area is of fundamental importance to calculate diverse physiological parameters such as the total rate of transpiration or photosynthesis of a plant or its leaf area index (LAI). This paper reports the determination of total leaf area in 3 year-old olive trees of two varieties using four different methods and leaf counting as the control method. Leaf area was determined on 7 plants of each variety, Arbequina and Cobrançosa, by thoroughly counting the total number of leaves on each plant and then multiplying by the average leaf area for each variety Leaf area of the same plants was then estimated by (1) the analysis, with software ImageJ, of a photo of each plant from a side or from the top, (2) the crown light scattering measured with a ceptometer (AccuPAR Model LP-80) in the ground at 10 and 20 cm from the trunk, (3) the analysis with software Hemisfer of a photo of the plant from the top, and finally (4) by the determination of the transpiration (mmol s-1) of each plant over a 2 hour period by a weighing method and dividing this value by the mean transpiration rate (E) of each plant, calculated from the measurement of stomatal conductance (gs) on 10 leaves with a porometer and mean air RH and leaf and air temperature over the same period. Both the ceptometer and the Hemisfer software give a value of leaf area index (LAI). In order to obtain the leaf area, LAI was divided by the projected area of the crown obtained on photos from the top. Average leaf area (n = 7 × 30) was 0,238 × 10-3 m2 and 0,264 × 10-3 m2 for Arbequina and Cobrançosa, respectively. Total leaf area of each plant varied from 3,38 m2 and 4,85 m2 for Arbequina and 0,86 m2 and 3,99 m2 for Cobrançosa. Ceptometer and analysis of photos from a side gave estimates of leaf area under 50% of the actual leaf area. Analysis of photos from the top gave on average 70% the actual values. The most accurate methods were analysis with software Hemisfer and by the ratio of transpiration by E which gave values 5% bellow or 20% above actual values, respectively