Development of an indirect ELISA against Equine Herpesvirus Types 1 and 4

The equine herpesviruses are important pathogens in the animal health context, namely in the equine’s health. So far, nine types have been identified, of which herpesvirus type 1 (EHV-1) and type 4 (EHV-4) are clinically, economically, and epidemiologically the most relevant due to the pathologies associated. The main objective of this work was to develop a peptide-based enzyme-linked immunosorbent assay (ELISA) for discrimination between EHV-1 and EHV-4. Selecting from the literature the immunogenic regions of both viruses, the tests were performed using a portion of glycoprotein E and a portion of glycoprotein G from each virus type. The results obtained until the conclusion of this work are promising, however, they were not conclusive. Nevertheless, they open the way for future testing, in order to make these ELISA test applicable for the detection of antibodies against EVH-1 and EHV-4.Este trabalho experimental teve como principal objetivo o desenvolvimento de um teste ELISA que permitisse detetar e diferenciar o herpesvírus equino tipo 1 (EHV-1) e o herpesvírus equino tipo 4 (EHV-4). Os herpesvírus equinos (EHVs) são vírus envelopados, com dupla cadeia de DNA que afetam todos os membros da família Equidae em todo o mundo (Wegdan et al., 2016; Slater, 2007), tendo sido, até ao momento, identificados e caracterizados nove tipos de herpesvírus. Os vírus dos tipos EHV-1, EHV-2, EHV-3, EHV-4 e EHV-5 infetam cavalos domésticos, enquanto os vírus dos tipos EHV-6, EHV-7, EHV-8 e EHV-9 estão associados a equídeos selvagens (Slater, 2007). Estes vírus possuem ciclos de vida complexos, que envolvem a infeção de múltiplos tipos celulares em diferentes tecidos e, são ainda detentores de diferentes mecanismos de evasão que lhes permite escapar à resposta imunológica do hospedeiro (Slater, 2007). Uma das ferramentas mais poderosas destes vírus é a capacidade de permanecer em latência, estado esse em que os equinos transportam o vírus de forma assintomática durante longos períodos. (Ostlound,1993; Balasuriya et al, 2015 Slater, 2007). Devido às suas características, os EHVs têm tido, a nível mundial e em todos os sectores da indústria equestre, um grande impacto económico e no bem-estar animal. (Slater, 2007; Milic et al., 2018). Contudo, de entre os EHVs, o EHV-1 e EHV-4 são do ponto de vista clínico, económico e epidemiológico os agentes patogénicos mais relevantes (Patel & Heldens, 2005; Slater, 2007). O EHV-1 é maioritariamente responsável por doenças respiratórias em animais jovens, aborto, morte neonatal e surtos extensivos de doença neurológica ou mielencefalopatia por herpesvírus equino (EHM), enquanto que o EHV-4 causa principalmente doenças respiratórias e raramente leva ao aborto (Ostlund, 1993; Balasuriya et al, 2015; Milic et al, 2018). Os vírus EHV-1 e EHV-4 foram em tempos considerados e classificados como dois subtipos do mesmo vírus (Milic et al., 2018). No entanto, a utilização da sequenciação genómica permitiu diferenciar estes dois vírus que são, contudo, geneticamente muito semelhantes (Vandekerckhove et al., 2011; Azab et al., 2012; Wegdan, 2016; Milic et al., 2018). Tendo em conta estas semelhanças entre os vírus, foram selecionadas para este trabalho, com base na literatura, duas regiões altamente imunogénicas que permitissem diferenciá-los sem que ocorresse reação cruzada. As regiões escolhidas foram uma porção do gene que codifica para a glicoproteína G (gG) e uma outra porção do gene que codifica para a glicoproteína E (gE). As glicoproteínas do herpesvírus estão presentes no envelope do vírus e têm papéis funcionais importantes na patogénese da doença (Crabb&Studdert,1990; Molinková,2012), mediando a entrada do vírus na célula hospedeira através da adsorção e penetração (Crabb & Studdert, 1990; Patel & Heldens, 2005; Slater, 2007). A maioria das diferenças entre EHV-1 e EHV-4 foram encontradas em 1993 por Crabb & Studdert na extremidade 3' do gene 70 que codifica a glicoproteína G. Comparando as sequências nucleotídicas de EHV-4 gG e EHV-1 gG, estes autores concluíram que as sequências possuíam 58% de identidade. No entanto, as regiões proteicas que incluíam os resíduos de aminoácidos 287 a 382 em EHV4 gG e 288 a 350 em EHV1 gG detinham apenas 21% de identidade. Por este motivo, esta região foi indicada como uma das mais promissoras na distinção de ambos os vírus. (Van Maanen, 2012; Thormann et al., 2012). Mais tarde, Damiani et al. (2000) examinaram mutantes de supressão do heterodímero gI/gE em EHV-4, em condições in vitro e in vivo, concluindo que os genes intactos destas glicoproteínas, gI e/ou gE, são necessários para uma transmissão eficaz do vírus através da propagação de célula a célula e, consequentemente, existe um papel importante dos genes gI e gE na virulência do EHV-4. Com base nestas descobertas, Andoh et al. (2012) identificaram e descreveram epítopos imunogénicos de EHV-1 e EHV-4 fundados em gE. Após o reconhecimento das regiões a utilizar, o presente trabalho foi desenhado de forma a obter estas proteínas recombinantes. Numa primeira fase, foram construídos dois vetores de expressão, cada um contento a porção imunogénica de gG de EHV-1 e EHV-4 para posterior transformação e expressão num organismo modelo neste caso, a bactéria Escherichia coli. E, em paralelo, o trabalho prosseguiu usando dois péptidos sintéticos de gE, um para cada um dos herpesvírus em estudo. A fase seguinte deste trabalho englobou o desenvolvimento de um teste ELISA, teste esse que tem como base reações antigénio-anticorpo detetáveis por meio de reações enzimáticas e têm sido utilizados para identificar estados de infeção e/ou vacinação de forma rápida e precisa. O ELISA desenvolvido foi do tipo indireto onde a ligação antigénio-anticorpo é detetada por um segundo anticorpo marcado o que confere uma maior especificidade e sensibilidade ao teste. No final, foi possível desenvolver e otimizar um teste ELISA indireto que permite a deteção de soros positivos e também a diferenciação de EHV-1 e EHV-4. No entanto, embora os resultados obtidos sejam promissores, trabalho futuro será necessário para testar um maior número de soros de forma a validar o teste ELISA e torná-lo apto para utilização em laboratório

Fast pattern recognition of malted and unmalted beer: An investigation using FTIR, UV-VIS, fluorescence spectroscopy and chemometrics

Beer production and consumption has increased worldwide during the past years. In this growing market, consumers have opted for products endowed with greater quality and diversity. In this respect, malted beers offer a more pleasant sensory experience. From a practical point of view, the high cost of production, when compared to the unmalted beer, passed on to the added value of the final product, then is common to encounter unmalted beers labeled as malted in the market. So, the characterization of beers into groups of malted and unmalted beers is of great importance for food control agencies. The present work reports a good alternative classification procedure that is fast, efficient, with no sample preparation using fluorescence spectroscopy associated with SOM (Self Organizing Map) and it is compared with the negative results (no pattern recognition) obtained with a FTIR and UV-VIS spectroscopy associated with PCA also performed with no sample preparation process

Different Lexicon-Based Approaches to Emotion Identification in Portuguese Tweets (Short Paper)

This paper presents the existing literature on the identification of emotions and describes various lexica-based approaches and translation strategies to identify emotions in Portuguese tweets. A dataset of tweets was manually annotated to evaluate our classifier and also to assess the difficulty of the task. A lexicon-based approach was used in order to classify the presence or absence of eight different emotions in a tweet. Different strategies have been applied to refine and improve an existing and widely used lexicon, by means of automatic machine translation and aligned word embeddings. We tested six different classification approaches, exploring different ways of directly applying resources available for English by means of different translation strategies. The achieved results suggest that a better performance can be obtained both by improving a lexicon and by directly translating tweets into English and then applying an existing English lexicon

Impacto da acidificação dos oceanos no desenvolvimento embrionário e larvar de peixes marinhos

Devido à acumulação atmosférica de CO2 antropogénico, a pressão parcial de dióxido de carbono (ρCO2) na água do mar aumenta e o pH diminui. Atualmente, este processo conhecido como a acidificação dos oceanos, está a ocorrer a um ritmo mais rápido do que em qualquer outro momento dos últimos 300 milhões de anos, o que leva a um desafio ecológico para os organismos marinhos a nível mundial. Embora este processo afete significativamente mais os organismos marinhos que sintetizam exosqueletos de calcário ou conchas, ainda não está claro como este processo irá afetar organismos calcificados internamente, tais como peixes marinhos. Embora se pense que os peixes adultos são relativamente mais tolerantes a níveis elevados de CO2 ambiental, sabe-se muito pouco sobre a sensibilidade dos estágios iniciais de vida, que são geralmente mais vulneráveis às mudanças ambientais do que os juvenis e os adultos. Determinar que espécies marinhas são sensíveis ou tolerantes aos elevados níveis de CO2 e redução do pH é fundamental para prever os impactos da acidificação dos oceanos nas cadeias alimentares marinhas e nos ecossistemas nos próximos 300 anos. O presente estudo aborda o efeito da acidificação dos oceanos sobre os estágios iniciais de desenvolvimento de três espécies comercialmente importantes, Solea senegalensis, Diplodus sargus, Argyrosomus regius. Estas espécies foram sujeitas a diferentes níveis de pH e ρCO2 (pH 8.0, ~400 μatm; pH 7.8, ~1000 μatm; pH 7.6, ~2000 μatm) desde a fase do ovo até à abertura de boca. Os resultados deste estudo sugerem que a exposição das fases iniciais de vida de Solea senegalensis e Diplodus sargus a elevadas concentrações de CO2 podem levar a taxas de eclosão e crescimento reduzidas e taxas de sobrevivência e peso seco elevados. Quando comparado com os níveis atuais de ρCO2 (400 ppm), a exposição de embriões Argyrosomus regius a 1000 μatm aumentou a sua sobrevivência, crescimento e comprimento total ao fim de 3 dias após eclosão. Não se detetaram diferenças significativas entre tratamentos no que respeita à organogénese e às dimensões e morfologia do ovo. De uma forma geral, este estudo parece indicar que Diplodus sargus e Solea senegalensis são substancialmente mais suscetíveis aos efeitos fisiológicos da acidificação dos oceanos do que Argyrososmus regius que está presumivelmente melhor adaptada às variações das condições ambientais, devido ao seu rápido desenvolvimento e ampla distribuição geográfica

Solar Irradiance (Global, Direct and Diffuse) Quality Control Methodologies. Review: Application to Time Series Measured At LES/LNEG, Lisboa, Portugal

ABSTRACT: Solar irradiance spatial and temporal quantification is essential to the development, implementation, and operation of solar systems, being used throughout a solar project lifecycle. It is crucial to have good quality data measured in meteorological and radiometric ground stations in order to enable the calibration and validation of irradiance models and data series. The Solar Energy laboratory at LNEG operates a meteorological station gathering relevant parameters to characterize the solar irradiation profile for the city of Lisbon in Portugal. This work presents and compares the application of different methodologies used for quality control of solar irradiance measurements. Three methods - the CIE (1994) / Muneer and Fairooz (2002), the QCRad and the IEC - were tested against two synthetic data sets: a clear-sky year and a typical meteorological year randomly and uniformly infused with errors. IEC showed to have limitation regarding the extreme value criteria for beam normal irradiance and CIE for the diffuse horizontal irradiance. The QCRad presented the best performance, with total sensitivity above 80% and maximum specificity. This method was applied to the measured data of LES-LNEG between 2014 and 2018. Most of the detected errors were detected during the coherence test stage, having a higher prevalence between 2015 and mid-2016, highlighting the need to modify the diffuse horizontal irradiance measuring system.info:eu-repo/semantics/publishedVersio

Fast estimation of the spectral optical properties of rabbit pancreas and pigment content analysis

The pancreas is a highly important organ, since it produces insulin and prevents the occurrence of diabetes. Although rare, pancreatic cancer is highly lethal, with a small life expectancy after being diagnosed. The pancreas is one of the organs less studied in the field of biophotonics. With the objective of acquiring information that can be used in the development of future applications to diagnose and treat pancreas diseases, the spectral optical properties of the rabbit pancreas were evaluated in a broad-spectral range, between 200 and 1000 nm. The method used to obtain such optical properties is simple, based almost on direct calculations from spectral measurements. The optical properties obtained show similar wavelength dependencies to the ones obtained for other tissues, but a further analysis on the spectral absorption coefficient showed that the pancreas tissues contain pigments, namely melanin, and lipofuscin. Using a simple calculation, it was possible to retrieve similar contents of these pigments from the absorption spectrum of the pancreas, which indicates that they accumulate in the same proportion as a result of the aging process. Such pigment accumulation was camouflaging the real contents of DNA, hemoglobin, and water, which were precisely evaluated after subtracting the pigment absorption

Cell adhesion in microchannel multiple constrictions - Evidence of mass transport limitations

Biofilm growth (fouling) in microdevices is a critical concern in several industrial, engineering and health applications, particularly in novel high-performance microdevices often designed with complex geometries, narrow regions and multiple headers. Unfortunately, on these devices, the regions with local high wall shear stresses (WSS) also show high local fouling rates. Several explanations have been put forward by the scientific community, including the effect of cell transport by Brownian motion on the adhesion rate. In this work, for the first time, both WSS and convection and Brownian diffusion effects on cell adhesion were evaluated along a microchannel with intercalate constriction and expansion zones designed to mimic the hydrodynamics of the human body and biomedical devices. Convection and Brownian diffusion effects were numerically studied using a steady-state convective-diffusion model (convection, diffusion and sedimentation). According to the numerical results, the convection and Brownian diffusion effects on cell adhesion are effectively more significant in regions with high WSS. Furthermore, a good agreement was observed between experimental and predicted local Sherwood numbers, particularly at the entrance and within the multiple constrictions. However, further mechanisms should be considered to accurately predict cell adhesion in the expansion zones. The described numerical approach can be used as a way to identify possible clogging zones in microchannels, and defining solutions, even before the construction of the prototype

Fast Estimation of the Spectral Optical Properties of Rabbit Pancreas and Pigment Content Analysis

The pancreas is a highly important organ, since it produces insulin and prevents the occurrence of diabetes. Although rare, pancreatic cancer is highly lethal, with a small life expectancy after being diagnosed. The pancreas is one of the organs less studied in the field of biophotonics. With the objective of acquiring information that can be used in the development of future applications to diagnose and treat pancreas diseases, the spectral optical properties of the rabbit pancreas were evaluated in a broad-spectral range, between 200 and 1000 nm. The method used to obtain such optical properties is simple, based almost on direct calculations from spectral measurements. The optical properties obtained show similar wavelength dependencies to the ones obtained for other tissues, but a further analysis on the spectral absorption coefficient showed that the pancreas tissues contain pigments, namely melanin, and lipofuscin. Using a simple calculation, it was possible to retrieve similar contents of these pigments from the absorption spectrum of the pancreas, which indicates that they accumulate in the same proportion as a result of the aging process. Such pigment accumulation was camouflaging the real contents of DNA, hemoglobin, and water, which were precisely evaluated after subtracting the pigment absorption.The work of I.S.M. was supported by the Portuguese Science Foundation, grant no. FCTUIDB/ 151528/2021. The work of H.F.S and L.M.O. was supported by the Portuguese Science Foundation, grant No. FCT-UIDB/04730/2020. The work of V.V.T. was supported by a grant under the Decree of the Government of the Russian Federation No. 220 of 09 April 2010 (Agreement No. 075-15-2021-615 of 4 June 2021).info:eu-repo/semantics/publishedVersio

Network process of children : influence of gender and type of game during cooperative-opposition games

The children, when playing, are communicating, since this is one of the forms of disinhibition and interaction with the group in which they are inserted (Lima, 2014). Ten children, five boys (n = 5; 5.8 ± 0.4 years old of age) and five girls (n = 5; 5.6 ± 0.5 years old of age) from a elementar school were observed during classes of motor expression. These classes took place once a week for a month, lasting one hour for each session. The interactions were observed with two cameras. It was counted as an interaction when a child passed one ball to another. The results suggest that it was in the “Free Game” that there were significant diferences between the type of game, since it was found in this one that it was easier for a player to be connected with his colleagues, being later the most requested, at the moment they had to pass the ball. Between the type of game and the gender, no significant interactions were found, as children who were the ones most requested by their classmates when they had to pass the ball were not verified. However, children who were crucial to maintaining cross-pass connections were also not checked. With regard to gender, no significant differences were found because no child, both male and female, has become important in sustaining the connections between the passes and the fact that no boys and / or girls were the most requested to make the passes with the ball. Finally, among the game types, no significant differences were found, since the children did not become essential to maintain the connection of passes between colleagues, in any of the matches played. The main objective of this study is to compare the interaction between female and male children in the type of cooperative-opposition games through Social Network Analysis.info:eu-repo/semantics/publishedVersio

Bee pollen nutritional value and microbiological stability: influence of preservation techniques

Bee pollen is an important beehive product, with a rich chemical composition and biological properties, which vary according to the region of production, plant age and agroecological conditions. Its moisture content may vary from 18 - 25%, depending on the technique and the time of collection [1]. Bee pollen is subject to proliferation of microbiological contamination that can make its consumption and commercialization unfeasible when not properly preserved, its nutritional value can be rapidly reduced due to Maillard reactions [2].info:eu-repo/semantics/publishedVersio