Detecting cells with low RNA content colonizing artworks non-invasively: RNA-FISH

Various non-invasive RNA-FISH methodologies were tested in this work. They seem to be good alternatives for analyzing the potential biodeteriogenic microorganisms thriving in CH objects.This work was co- financed by FCT Fundação para a Ciência e a Tecnologia through the project "MICROTECH-ART- Microorganisms Thriving on and Endamaging Cultural Heritage -an Analytical Rapid Tool-" (PTDC/BBB-IMG/0046/2014) and by European Union, European Regional Development Fund ALENTEJO 2020 through the project HIT3CH - HERCULES Interface for Technology Transfer and Teaming in Cultural Heritage (ALT20-03-0246-FEDER-000004). Marina González-Pérez acknowledges FCT for the economic support through the post-doctoral grant SFRH/BPD/100754/2014

Species-specific RNA-FISH probes for yeast identification: evaluation of their specificity and performance

The aim of this work was to investigate the possibility of applying the RNA-FISH protocol previously developed by our group for simultaneous analysis of fungi and bacteria using kingdom-specific probes for specific detection of various yeast species using previously published species-specific probes. Their specificity and performance were analysed both in silico and experimentally.FCT through PTDC/BBB-IMG/0046/2014 project and SFRH/BPD/100754/2014 grant and ALT20-03-0246-FEDER-000004-ALENTEJO 2020 projec

Overcoming the limitations to identify Dekkera bruxellensis in wine environment: performance and specificity evaluation of two species-specific RNA-FISH probes.

RNA-Fluorescence In Situ Hybridisation (RNA-FISH) allows the detection and identification of microorganisms in complex matrices in few hours being considered as one of the most powerful techniques for various applications in microbiology. Thus, it has the analytical potential for allowing specific detection of D. bruxellensis. However, some experimental difficulties can hamper its successful application such as: a) the autofluorescence of the sample and its interference with the RNA-FISH probe-conferred fluorescence, and b) the lack of probes with the desired levels of specificity and FISH performance. This is why this study was focused on overcoming the possible experimental drawbacks that can be found in RNA-FISH application for detecting D. bruxellensis in the wine environment and on evaluating the performance and specificity (both in silico and experimentally) of two RNA-FISH probes targeting to D. bruxellensis: a novel probe designed by us and a probe previously designed by other authors, 26S D. brux.5.1 .This work was co-financed by Foundation for Science and Technology (FCT) through the project PTDC/BBB-IMG/0046/2014 and the post-doctoral grant SFRH/BPD/100754/2014 and by ALT20-03-0145-FEDER- 000015 project

Fluorophore´s role on the reliability of microorganism detection by Fluorescence In Situ Hybridisation (FISH).

FISH has been applied in many ecological and phylogenetic studies becoming the method of choice for the direct detection and identification of microorganisms in their natural environments [1]. For reliable results, it is crucial to minimise or avoid background fluorescence and cellular autofluorescence but also to maximise the specific FISH signals obtained. Red-emitting dyes have been usually used for eliminating the problems of non-specific fluorescence interference. Therefore, in this work we evaluated the role of three of these fluorophores (Alexa Fluor 647 (AF647), ATTO 647N and Cy5) on the reliability of the RNAFISH results obtained both with a universal and a specie-specific probe.ALT20-03-0246-FEDER-000004-ALENTEJO 2020 project and FCT through PTDC/BBB-IMG/0046/2014 project and SFRH/BPD/100754/2014 grant

Separation of phthalates by cyclodextrin modified micellar electrokinetic chromatography. Quantitation in perfumes.

A new CE method has been developed for the simultaneous separation of a group of parent phthalates. Due to the neutral character of these compounds, the addition of several bile salts as surfactants (sodium cholate (SC), sodium deoxycholate (SDC), sodium taurodeoxycholate (STDC), sodium taurocholate (STC)) to the separation buffer was explored showing the high potential of SDC as pseudostationary phase. However, the resolution of all the phthalates was not achieved when employing only this bile salt as additive, being necessary the addition of neutral cyclodextrins (CD) and organic modifiers to the separation media. The optimized cyclodextrin modified micellar electrokinetic chromatography (CD-MEKC) method consisted of the employ of a background electrolyte (BGE) containing 25 mM beta-CD-100 mM SDC in a 100 mM borate buffer (pH 8.5) with a 10% (v/v) of acetonitrile, employing a voltage of 30 kV and a temperature of 25 degrees C. This separation medium enabled the total resolution of eight compounds and the partial resolution of two of the analytes, di-n-octyl phthalate (DNOP) and diethyl hexyl phthalate (DEHP) (Rs similar to 0.8), in only 12 min. The analytical characteristics of the developed method were studied showing their suitability for the determination of these compounds in commercial perfumes. In all the analyzed perfumes the most common phthalate was diethyl phthalate (DEP) that appeared in ten of the fifteen analyzed products. Also dimethyl phthalate (DMP), diallyl phthalate (DAP), dicyclohexyl phthalate (DCP), and di-n-pentyl phthalate (DNPP) were found in some of the analyzed samples. (C) 2013 Elsevier B.V. All rights reserved

RNA-FISH for investigating Cultural Heritage Microbiology- Autofluorescence of the materials and fluorophores selection.

This work was focused on: i) the investigation of the autofluorescence of various materials that are found forming part of Cultural Heritage objects (parchment, paper, wood, fabric, rock, mortar and plastic among others) by epifluorescence microscopy using fluorescence flter sets targeted at commonly used fluorophores (TRITC/CY5/6-FAM); ii) the detection of RNA-FISH stained microbial cells on the selected materials or in the presence of residual particles.This work was co-financed by FCT – Fundação para a Ciência e a Tecnologia through the project "MICROTECH-ART- Microorganisms Thriving on and Endamaging Cultural Heritage -an Analytical Rapid Tool-" (PTDC/BBB-IMG/0046/2014) and post-doctoral grant SFRH/BPD/100754/2014 and by European Union, European Regional Development Fund ALENTEJO 2020 through the project “HIT3CH - HERCULES Interface for Technology Transfer and Teaming in Cultural Heritage” (ALT20-03-0246-FEDER-000004)

detection for yeast and bacteria in wood slabs by RNA FISH

The deterioration of cultural heritage objects and assets (mural paintings, statues, and many other art objects made of wood, stone, paper, ceramic, glass, inter alia) can be caused by microorganisms [1]. One of the most important steps for applying adequate conservation and protection measures is early identification and monitorization of microbial colonization. The conventional culture-based methods used so far have become insufficient to detect/identify the biodeteriogenic agents. Thus, molecular techniques have started to attract considerable interest [2,3]. Our group is focused on detecting and identifying microorganisms that cause biodeterioration on artworks using the RNA-FISH molecular technique [4]. It is a simple, rapid and promising molecular technique enabling the detection, visualization and identification of the viable microorganisms of interest [5,6]. As any other technique, RNA-FISH has its own minimum Limit Of Detection (LOD) and for ensuring the reliability of RNA-FISH analyses, determination of the associated LODs is imperative. Thus, the aim of this work was to determine the LOD for yeast and bacteria in wood slabs by RNA-FISH. Universal probes for targeting eukaryotes (EUK516) and bacteria (EUB338) labeled with Cy3 or Atto-647N dyes were used.This work was co-financed by ALT20-03-0246-FEDER-000004-ALENTEJO 2020 project and by FCT through the project PTDC/BBB-IMG/0046/2014 and grants SFRH/BD/118028/2016 and SFRH/BPD/100754/2014

Propuesta didáctica para la prevención del acoso escolar en las aulas de educación primaria.

Este Trabajo de Fin de Grado, se presenta como propuesta didáctica enfocada a la prevención del acoso escolar en las aulas de Educación Primaria. Comenzamos realizando un estudio acerca del acoso escolar infantil a diferentes niveles (Mundial, Europa, España y Canarias). Posteriormente, se profundizará sobre las actitudes que se desarrollan sobre los iguales y los aislamientos a diferentes escolares en un centro de Educación Primaria a través de un cuestionario. Trasla obtención de los resultados, se observará la necesidad de un cambio. Partiendo de los datos obtenidos, se planteará una propuesta didáctica orientada a: mejorar las deficiencias que se han encontrado, prevenir las actitudes negativas sobre los iguales, sensibilizar a los docentes y familias de la importancia de educar implicando la comunicación asertiva y, sobre todo, fomentar la empatíaIn this Degree´s Final Project we will carry out a study on child bullying at different levels (Universal, Europe, Spain and the Canary Islands). Then, we will delve into the attitudes that are developed about peers and the isolations to different school children in a Primary Education center through a questionnaire. After obtaining the results, the need for a change will be observed. A didactic proposal will be proposed aimed at: improving the deficiencies that have been found, preventing negative attitudes about peers, raising awareness among teachers and families of the importance of educating involving assertive communication, and, above all, promoting empathy. Finally, we will present a didactic proposal aimed at: improving the deficiencies found, preventing negative attitudes about peers, creating awareness among teachers and families about the importance of educating with assertive communication and, above all, promoting empathy

San Pedro Martir observations of microvariability in obscured quasars

Fast brightness variations are a unique tool to probe the innermost regions of active galactic nuclei (AGN). These variations are called microvariability or intra-night variability, and this phenomenon has been monitored in samples of blazars and unobscured AGNs. Detecting optical microvariations in targets hidden by the obscuring torus is a challenging task because the region responsible for the variations is hidden from our sight. However, there have been reports of fast variations in obscured Seyfert galaxies in X-rays, which rises the question whether microvariations can also be detected in obscured AGNs in the optical regime. Because the expected variations are very small and can easily be lost within the noise, the analysis requires a statistical approach. We report the use of a one-way analysis of variance, ANOVA, with which we searched for microvariability. ANOVA was successfully employed in previous studies of unobscured AGNs. As a result, we found microvariable events during three observing blocks: in two we observed the same object (Mrk 477), and in another, J0759+5050. The results on Mrk 477 confirm previous findings. However, since Mrk 477 is quite a peculiar target with hidden broad-line regions, we cannot rule out the possibility that we have serendipitously chosen a target prone to variations.Comment: Research note, 5 pages, 2 figures, accepted for publication in Astronomy and Astrophysic

Forage quality of consecutive years interact to affect body condition, reproductive rate and rut phenology in Iberian red deer

Body condition for reproduction in capital breeders such as the red deer (Cervus elaphus) is mostly determined by their stored energy reserves. Thus, environmental conditions and resource availability may affect reproductive performance and breeding success. In warm Mediterranean regions, current climate change is driving to a hotter and drier scenario that is expected to affect the biology and dynamics of many populations. We examined the impact of these local climate variations on red deer body condition and the relationship with female reproductive phenology and breeding success. We used satellite information of landscape vegetation along with a 22-year data series of direct field behavioural observations during the rutting season in Doñana National Park (SW Spain). We analyzed faecal nitrogen content (FN) from faeces collected during the rut. We found that poor vegetation availability in drier years was related to worse body condition of deer (measured by FN) and a delay in the rutting season, which associated with lower reproductive rates (measured by the proportion of females with calves observed the next year). We also evidenced an interesting interaction between environmental conditions in consecutive years on the timing of breeding season, with timing of breeding being more delayed when previous year resource availability was high and many females bred, and the consecutive one was poor, so females hardly recovered condition and the rut occurred later. These findings highlight the carry-over effect of reproduction in capital breeders and the potential impact of climate-change conditions on red deer breeding