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© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)., Students with chronic conditions (CC) tend to experience several barriers in terms of their school participation and performance. Therefore, the present study aims to explore the factors related to the time of diagnosis of CC (recent/non-recent), the barriers to participation and academic success (health condition, people's attitude towards CC and school physical environment), the physical and mental health (physical/psychological symptoms and concerns) and school-related variables (relationship with teachers and peers), regarding the school participation of first-year students with CC. This work is part of the HOUSE-Colégio F3 Project, University of Lisbon, which includes 1143 first-year university students from 17 Faculties and Institutes of the University of Lisbon. In this specific study, only the subsample of 207 students with CC was considered, 72.4% of which were female, aged between 18 and 54 years (M = 20.00; SD = 4.83). The results showed that students with a recent diagnosis of CC and students with school participation affected by the CC were those who presented more negative indicators regarding barriers to school participation, physical and mental health, and school-related variables. A greater impact of CC in terms of school participation was associated with having a recent diagnosis, with people's attitude towards CC and with the health condition as barriers, with more psychological symptoms and worse relationships with teachers and peers. This is a relevant message for the organization of health services for students with CC at the beginning of their university studies, especially since they are often displaced from home and managing their health conditions alone (in many cases, for the first time)., Ana Cerqueira—Foundation for Science and Technology (FCT) Grant (SFRH/BD/148403/2019). Fábio Botelho Guedes—Foundation for Science and Technology (FCT) Grant (SFRH/BD/148299/2019).

Stressful high temperatures on plants can limit whole-plant function and decrease crop productivity. However, little is known regarding heat stress effects on microtubule cytoskeleton and chromatin in roots from intact plants. Here we studied high temperature effects on cell division, microtubule and chromatin organization patterns in rye root tips from intact plants subjected to 40ºC for 4 h and after different recovery periods (0RT, 7RT, 24 RT). We showed that heat stress induced changes in nuclear morphology as detected by the unusual presence of interphase cells with irregularly shaped nuclei, probably associated with changes in chromosome segregation at anaphase, leading to micronuclei formation as well as changes in the mitotic index. These alterations were associated to differential effects in microtubules organization in both heat-stressed interphase and mitotic cells at 0RT and 7RT. Although no changes in the distribution of H3 phosphorylation of Ser 10 residues on chromatin were found in cells from heat-stressed plants, marked alterations in chromatin DNA methylation patterns were detected. These effects included higher agglutination of 5-methylcytosine domains in both interphase and metaphase cells compared to controls. Taken together these results seem to suggest that alterations in microtubule conformation upon heat stress influences nuclear chromatin organization and cell cycle progression. However, when seedlings recovered from stress (24RT), root tip cells presented microtubule configurations and chromatin organization patterns similar to controls. We conclude that in spite of heat stress markedly altered cell cycle progression and distribution of epigenetic marks, these responses are transient to cope with such stress conditions in the roots., Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics, Just Accepted

Grapevines are highly dependent on arbuscular mycorrhizal fungi (AMF) for normal growth and development. However, vineyard soils may have low AMF abundance and diversity due to conventional soil management practices that are detrimental for these fungi. In this context, the establishment of AMF-inoculated cover crops can be a highly convenient strategy to reestablish soil mycorrhizal potential, as it combines the advantages of a vigorous inoculum source coming from mycorrhizal donor plants with the overall benefits of green covers for grape quality, microbial diversity and soil health. In this work, the potential benefits of Funneliformis mosseae-inoculated under-vine cover crops on grapevine growth, physiology and production were compared to those derived from 1) the establishment of non-inoculated under-vine cover crops, and 2) conventional herbicide-based weed control in the under-vine space. In addition, grapevine root AMF community composition was analyzed to assess if the introduction of a non-native AMF species induced changes on resident mycorrhizal community assemblies and to unveil potential variations in AMF diversity associated to herbicide replacement by green covers. Results indicated that under-vine cover crops, inoculated or not, led to a general vigor decrease in grapevines, probably due to competition between the two species. However, after a heat wave that occurred at harvest time in the second year of the experiment, grapevines growing in plots with inoculated cover crops had the highest photochemical reflectance indices and net photosynthesis rates, and partially compensated production losses due to berry sunburn. Root mycorrhizal community analysis by the end of the experiment revealed that the inoculated F. mosseae isolate colonized grapevine roots from inoculated plots, while it was absent in the other ones. Moreover, inoculation of this AMF did not lead to a replacement of native root AMF communities, but allowed further colonization by other resident Glomeraceae and non-Glomeraceae AMF taxa. Overall, the work herein demonstrates that the introduction of F. mosseae through donor plants is a suitable field inoculation method for grapevines and can help them to better withstand heat waves info:eu-repo/semantics/submittedVersion

Continuous application of Cu-based fungicides and molluscicides may lead to Cu accumulation in vineyard soils. Grapevine inoculation with arbuscular mycorrhizal fungi (AMF) can be a suitable strategy to increase plant tolerance to the excessive levels of this element in the soil. However, the simultaneous use of EDTA-based fertilizers or phytosanitary products in those vineyards can affect metal mobility in the soil, with potential negative consequences for plants. Therefore, this study aimed to elucidate the effect of AMF inoculation on grapevine performance in Cu contaminated soils, in the presence or absence of EDTA chelates. Two-year-old plants growing in pots filled with an Arenosol, previously inoculated or not with two different AMF, were subjected to three soil treatments: control, Cu addition; Cu and FeNaEDTA addition. Four months later, at the end of the growing season, plant vegetative growth parameters, root colonization as well as soil, root and leaf nutrient contents were analyzed. In the soil where Cu was applied, Rhizoglomus irregulare- and Funneliformis mosseae-inoculated plants showed significantly higher root biomass than the non-inoculated ones, and R. irregulare-inoculated plants were the ones with the highest root P and Fe concentrations. However, when Cu and FeNaEDTA were applied together to the soil, AMF inoculation was no longer an asset for grapevines, and overall, root biomass decreased, translocation of Cu to leaves was stimulated, and a significant decrease in leaf Mn concentrations was detected, below the levels found in control soils. For this reason, in soils with high Cu-concentrations, although mycorrhizal inoculation can be a suitable strategy to improve plant growth and nutrition, the use of EDTA-based products should be avoided, as it can lead to an amplification of the toxic effects commonly caused by the excess of Cu in plants info:eu-repo/semantics/submittedVersion

Major depressive disorder is a moderately heritable disorder characterized by one or more major depressive episodes. Laboratory tests to suport MDD diagnosis are not available. Diagnosis and treatment are based on various signs and symptoms not always fitting into strict diagnostic categories. Research for biological markers of neuropsychiatric disorders has been a challenge.

O Bisphenol A (BPA) é um xenoestrógeneo sintético capaz de ativar diversas vias de sinalização associadas a estrogénios. Este composto é utilizado na produção de plástico e das resinas de cola Epoxy, encontrando-se presente numa grande variedade de produtos para consumo humano. Consequentemente a exposição humana ao BPA é considerada generalizada e contínua. Estudos epidemiológicos verificaram correlação entre níveis elevados de BPA na urina e patogénese de doenças associadas ao envelhecimento celular como a aterosclerose. Neste estudo foram avaliados os efeitos da exposição prolongada ao BPA de concentrações encontradas em amostras biológicas humanas associadas a exposição ambiental e ocupacional, nomeadamente 10 ng/mL e 1 μg/ml.

Trabajo presentado en la V Meeting Asociación Española de Leguminosas, AEL (Eucarpia International Symposium on Protein Crops), celebrada en Pontevedra del 4 al 7 de mayo de 2015., Faba bean (Vicia faba L.) is an important crop because it is a legume with seeds that are a very relevant source of protein for human and animal consumption. It is an entomogamous partially allogamous crop that recently has been considered a candidate for low input and organic agriculture. So, the characterization of ex situ and on farm germplasm has a stronger importance for this crop. The aim of this work was to estimate the diversity within and among locally adapted faba bean accessions using the seeds morphological characteristics and the Inter-retrotransposon Amplification Polymorphism (IRAP) technique. We have studied four Portuguese accessions adapted to different agroecological conditions (two accessions of the major group and other two of the equina group) and, for control purposes, two inbred lines from CSIC belonging to the equina group. The plants were grown under pollinator-exclusion environment. The morphological characters analyzed were the number of seeds per pod, the 100 seed weight and the seed length, width and thickness. IRAP profiles were obtained using a primer for LTR regions of Athila retrotransposon. Cluster analysis using UPGMA and Principal Component Analysis (PCA) were performed to examine the variation among the studied accessions and to identify seed characters more relevant to distinguish the accessions analyzed. For the IRAP data a cluster analysis was also used and a Principal Coordinate Analysis (PCoordA) was performed. The cluster analysis of the morphological data combined with the PCA placed major and equina accessions into separated groups. PCA application moreover revealed that the main characters discriminating botanical groups were 100 seed weight and seed dimensions. Concerning the IRAP results, both the cluster analysis using UPGMA and the PCoordA separated the studied accessions in four groups. The two accessions of major faba bean formed one group quite distinct from all the equina accessions. Among the equina accessions the following three groups can be considered: (i) one group is formed by a Portuguese landrace presenting the highest degree of intravariation, which can be related to the great morphologic diversity existing in this accession; (ii) a second group formed by one inbred accession and (iii) a third group composed by one locally adapted equina Portuguese accession and the other inbred line. This study shows that seed morphological traits and the IRAP marker give similar results in what concerns the discrimination of faba bean major and equine groups. The IRAP marker could be a good choice to deeper assess genetic diversity in faba bean germplasm.

The functional importance of Epigenetics arise from DNA sequencing programs that show the need for another code to explain the dynamics of gene expression patterns observed along cell differentiation and organism development. In this context, the study of ribosomal gene silencing is in fact an excellent model to better understand the relationships that are established between gene transcription and chromatin topology, and to unravel the epigenetic switches evolved in the framework of gene expression.

Allopolyploidy is a significant evolutionary process, resulting in new species with diploid or greater chromosome complements derived from two or more progenitor species. We examined the chromosomal consequences of genomic merger in Arabidopsis suecica, the allotetraploid hybrid of Arabidopsis thaliana and Arabidopsis arenosa. Fluorescence in situ hybridization with centromere, nucleolus organizer region (NOR), and 5S rRNA gene probes reveals the expected numbers of progenitor chromosomes in natural A. suecica, but one pair of A. thaliana NORs and one pair of A. arenosa-derived 5S gene loci are missing. Similarly, in newly formed synthetic A. suecica-like allotetraploids, pairs of A. thaliana NORs are gained de novo, lost, and or transposed to A. arenosa chromosomes, with genotypic differences apparent between F3 siblings of the same F2 parent and between independent lines. Likewise, pairs of A. arenosa 5S genes are lost and novel linkages between 5S loci and NORs arise in synthetic allotetraploids. By contrast, the expected numbers of A. arenosa-derived NORs and A. thaliana-derived 5S loci are found in both natural and synthetic A. suecica. Collectively, these observations suggest that some, but not all, loci are unstable in newly formed A. suecica allotetraploids and can participate in a variety of alternative rearrangements, some of which resemble chromosomal changes found in nature.

In genetic hybrids, nucleolus formation on chromosomes inherited from only one parent is the epigenetic phenomenon, nucleolar dominance. By using Arabidopsis suecica, the allotetraploid hybrid of Arabidopsis thaliana and Arabidopsis arenosa, natural variation in nucleolar dominance was found to occur, providing a unique opportunity to examine homologous nucleolus organizer regions (NORs) in their active and inactive states. In A. suecica strain LC1, NORs derived from A. arenosa are active, whereas A. thalianaderived NORs are silenced. In A. suecica strain 9502, NORs of both parental species are active. When active, NORs are partially, but not fully, decondensed. Both active and inactive LC1 NORs colocalize with the nucleolus, contradicting the long-standing assumption that rRNA gene transcription drives nucleolus association. Collectively, these observations clarify the relationships among NOR chromatin topology, rRNA gene transcription, and NOR–nucleolus associations. A. suecica strains LC1 and 9502 have each lost one pair of A. thaliana NORs during evolution, and amplified fragmentlength polymorphism analysis further indicates that these strains are genetically very similar. These data suggest that nucleolar dominance can result from subtle genetic or epigenetic variation but is not a trait fundamental to a given interspecies hybrid combination

Nucleolar dominance is an enigma. The puzzle of differential amphiplasty has remained unresolved since it was first recognised and described in Crepis hybrids by Navashin in 1934. Here we review the body of knowledge that has grown out of the many models that have tried to find the genetic basis for differential rRNA gene expression in hybrids, and present a new interpretation. We propose and discuss a chromatin imprinting model which re-interprets differential amphiplasty in terms of two genomes of differing size occupying a common space within the nucleus, and with heterochromatin as a key player in the scenario. Difference in size between two parental genomes induces an inherited epigenetic mark in the hybrid that allows patterns of chromatin organization to have positional effects on the neighbouring domains. This chromatin imprinting model can be also used to explain complex genomic interactions which transcend nucleolar dominance and which can account for the overall characteristics of hybrids. Gene expression in hybrids, relative to parentage, is seen as being based on the nuclear location of the sequences concerned within their genomic environment, and where the presence of particular repetitive DNA sequences are ‘sensed’, and render silent the adjacent information.

[EN] Through the use of a silver staining technique three distinct regions can always be visualized in chromosomes:nucleolus organizer regions, centromere and telomere regions. The organization of the centromere region was studied in different phases of mitosis and meiosis. The centromere region was shown to be formed by an assembled dot-like structure with a market different complexity between mitotic and metaphase I chromosomes, being quadripartite in the former and bipartite in the latter. The nature of this difference is suggested to be related to the mechanism responsible to reductional division. The protein nature of these dots and their correspondence to the chromosome "core" and to the "scaffolding" is discussed. The presence of terminal dots in almost all telomeres and the existence of intercalar dots in some chromosomes are interpreted as being dependent on the individual phylogeny for each chromosome., [ES] Utilizando la técnica de tinción por el nitrato de plata, fue posible visualizar tres importantes regiones de los cromosomas de trigo panadero y de centeno: la centromérica, la telomérica y la del organizador nucleolar. Se observó que la región centrométrica era cosntituída por una estructura puntillada y densamente teñida cuya complejidad dependía de que los cromosomas estuviesen en la metafasis de mitosis o en la de la primera division de meiosis. En la última, la estructura del centrómetro era bipartida (Fig. 1A) mientras que en la primera se presentaba quadripartida (Fig. 1C, D). Se sugiere que esta estructura meiótica de los centrómeros pueda relacionarse con la división reducional que se sigue. Efectivamente los centrómeros son quadripartidos en la metafase ecuacional siguiente. Se discute la relación entre la constitución protéica de la estructura centromérica y los "core" y el "scaffolding" cromosómicos estudiados por varios autores. También se discute el origen de los puntos teloméricos e intercalares observados en los cromosomas cuando están teñidos por esta técnica.

Doutoramento em Biologia / Instituto Superior de Agronomia. Universidade de Lisboa Wheat is an essential crop for food and feed, due to its nutritional value and unique aptitude to produce gluten and their derived food products. It is thus essential to understand how increasingly common extreme weather events like heatwaves, defined as short periods of high temperatures (HT), affect wheat grain production and quality, and transcriptomic modulation. Thus, in order to identify wheat varieties with increased tolerance to HT, the objective of this work focused on the characterization of the already referred parameters in plants of several commercial varieties recommended to be used nowadays in Portugal and traditional varieties, submitted to high temperatures during grain filling. Using molecular markers, we showed that commercial genotypes have predicted good grain technological quality, based on the allelic composition of genes related with grain composition. Most commercial and traditional genotypes showed negative effects, induced by heatwave-like treatment, revealed by a decrease in grain number and weight, while protein content was increased. Also, through attenuated total reflection Fourier transform infrared (ATR-FTIR) analysis, we denoted the occurrence of alterations in grain polysaccharide composition induced by HT. Additionally, HT increased protein content variability in landraces and reduced it on commercial varieties. Regarding transcriptomic profiles assessed immediately after the HT treatment, traditional varieties revealed a significantly higher number of differentially expressed genes (DEGs), that include genes coding for heat shock proteins and cupins, and more similar HT responses than commercial varieties. Furthermore, Bancal and landraces DEGs appear to be more associated with several metabolic pathways, while in Antequera DEGs were preferentially related with transcription modulation and RNA and protein synthesis N/A

Mestrado em Engenharia Agronómica - Instituto Superior de Agronomia Glycoproteins are a major part of biopharmaceutical products. For their production, a high level of homogeny is required, not only for the protein but also for attached glycans, since these oligossacharides influence protein function. For this goal, plants emerge as an ideal biopharmaceutical production platform. By infiltrating adult plant leaves with a suspension of Agrobacterium tumefaciens containing a plasmid for the gene of interest, a high amount of protein can be produced in a short period. In this study, the production of two antibodies in genetically glycoengineered Nicotiana benthamiana was described. These antibodies, Rituximab and KBPA, served as models for Immunoglobulin groups G and M. Additionally, co-infiltration of the genes for these antibodies with glycoengineering constructs allowed for specific glycan structures attached to these proteins. Finally, the down-regulation of β-N-acetylhexosaminidase 3 with RNAi technology was described, inhibiting the effect of this glycosidase on plant produced proteins.

Doutoramento em Biologia - Instituto Superior de Agronomia Bisphenol A (BPA) is an extensively utilized endocrine disruptor for which human exposure is considered generalized through ingestion. Information regarding BPA effects on vascular and digestive tract tissues is scarce. Therefore, in this work primary Human Umbilical Vein Endothelial Cells (HUVEC) and human colon adenocarcinona cell line HT29 were used to evaluate BPA effects at two distinct low-dose concentrations relevant in terms of human health risk assessment. BPA differentially affects the cell types studied, with more pronounced aneugenic effects, nucleolar disruption and transcriptional deregulation observed in HUVEC. Prolonged BPA exposure affects aging processes in senescent HUVEC. Interaction experiments involving expression of key cancer related genes shows that BPA antagonizes transcriptional effects of the chemotherapeutic agent doxorubicin in HT29. Additionally BPA aneugenic effects are enhanced by co-exposure with Eupatorium cannabinum L. ethanolic extract, a medicinal plant, for which a potent cytotoxic activity against HT29 cells is also demonstrated here. Altogether these results support increasing concerns regarding harmful effects of BPA at low-dose on human health and draw attention to the importance of a deeper understanding of BPA potential interactions with other chemicals.

Doutoramento em Biologia - Instituto Superior de Agronomia Polyploidization is a remarkable driving force in plant evolution, where hybridization and chromosome doubling result in huge genomic stress. Deeper knowledge about parental genomes behaviour in a hybrid nucleus and on processes underlying genetic and epigenetic modifications induced by polyploidization is essential to understand such evolutive process. Using triticale as model species we evaluated the impact of polyploidization through molecular and cytological approaches. Genomic rearrangements involving retrotransposons and microsatellites related sequences include both repetitive and coding sequences, and revealed a preferential loss of rye-origin bands. Chromosome distribution of such marker sequences demonstrated moreover enrichment in heterochromatic domains. Intensification of rye genome rearrangements was also disclosed in wheat lines with disomic additions of rye chromosomes, probably due to unbalanced genomic constitution. An integrative review of genomic modulation evaluated in Triticeae hybrid/polyploidy species unravelled furthermore higher restructuring of larger parental genomes, emphasizing the importance of genome size homogenization. Epigenetic analysis of nucleolar dominance in wheat addition line with rye nucleolar chromosomes revealed an unexpected up-regulation of ribosomal genes from wheat-origin, suggesting that mutual and opposite expression patterns modifications are induced by genome interactions. Altogether we demonstrate that heterochromatic domains are highly involved in parental genomes adjustments required to polyploids stabilization.--------------------------------------------A poliploidização é uma das principais forças evolutivas em plantas, sendo essencial um conhecimento profundo dos processos subjacentes às alterações genómicas e epigenéticas associadas a este processo evolutivo. O triticale foi utilizado como espécie modelo para avaliar o impacto da poliploidização utilizando técnicas moleculares e citológicas. Identificaram-se rearranjos genómicos envolvendo sequências repetitivas e codificantes associadas a retrotransposões e microssatélites, afectando preferencialmente o genoma de centeio. A distribuição cromossómica dessas sequências demonstrou a sua predominância em domínios heterocromáticos. Paralelamente, em linhas de trigo com a adição dos cromossomas de centeio, observou-se uma intensificação de rearranjos nesse genoma, provavelmente resultante do desequilíbrio genómico parental. A integração dos estudos realizados em híbridos/poliplóides pertencentes à tribo Triticeae demonstrou a reestruturação preferencial do genoma parental maior, realçando a importância da homogeneização genómica. A análise epigenética dos genes ribossomais na linha de trigo com introgressão de cromossomas nucleolares de centeio, indica que o processo de dominância nucleolar induz modificações mútuas dos padrões de expressão genica dos progenitores. Este trabalho enfatiza assim a importância da modulação dos domínios heterocromáticos parentais na estabilização dos organismos poliploides

Doutoramento em Biologia - Instituto Superior de Agronomia Submitted by Margarida Galamba (galamba@isa.utl.pt) on 2012-02-09T14:46:27Z No. of bitstreams: 1 REP-Doutoramento-Bento M PhD 2011.pdf: 7622021 bytes, checksum: 07370571d7a0afed2e70caced501a522 (MD5) Made available in DSpace on 2012-02-09T14:48:40Z (GMT). No. of bitstreams: 1 REP-Doutoramento-Bento M PhD 2011.pdf: 7622021 bytes, checksum: 07370571d7a0afed2e70caced501a522 (MD5) Previous issue date: 2011

Mestrado em Biologia Funcional - Instituto Superior de Agronomia The aim of this work was to evaluate the influence of high temperature stress (HTS) in the chromatin organization of two repetitive sequences – 45S ribossomal DNA and subtelomeric pSc200 – and of nucleoli in interphase root tips and coleoptiles nuclei from Secale cereale seedlings using fluorescent in situ hybridization and imunocitochemestry, respectively. Furthermore we analyzed the influence of high temperature stress in the transcription patterns of the same sequences through quantitative real-time PCR. Nuclei from both organs show different conformation patters. Root tips nuclei have bigger dimensions, more disperse subtelomeric FISH signals and more compact ribossomal probe. We observe that high temperature stress causes chromatin decondensation in both sequences in root tips nuclei, while in coleoptile nuclei, beside the rDNA decondensation, we observe an increase in the nuclear area suggesting a global nuclear decompactation. The nucleolar area is also higher in both types of nuclei of stressed seedlings. This increase could be correlated with the increase in the ribossomal genes expression level induced by temperature stress, demonstrated through real-time PCR. The present work describes for the first time that the subtelomeric sequence pSc200 is transcribed, although its levels are not significantly altered with high temperature stress.

Doutoramento em Biologia - Instituto Superior de Agronomia The discovery of small RNAs has added a new level to our understanding of gene regulation mechanisms. Small interfering RNAs (siRNAs) are involved in the establishment of repressive epigenetic marks in a variety of organisms by guiding RNA induced silencing complexes to homologous DNA sequences. Could the endogenous siRNA heterochromatic pathway be linked to the establishment of nucleolar dominance? The latter is a large scale manifestation of directed epigenetic gene silencing, resulting in the transcriptional inactivation of the entire rRNA gene cluster from one parent in a hybrid species. Inactivation of rRNA genes is linked to the establishment of heterochromatic marks, both at the DNA and histone levels, in the promoter region. We were able to identify 24nt siRNAs homologous to the rRNA gene promoter in Arabidopsis sp., whose biogenesis is dependent on nuclear RNA polymerase IV and other known proteins of the endogenous siRNA pathway in Arabidopsis. RNAi mediated knockdown of genes in this pathway in the natural allopolyploid hybrid A. suecica (A. thaliana x A. arenosa) disrupted nucleolar dominance, causing the production of rRNA transcripts from the underdominant A. thaliana rDNA genes. This observation implicates nuclear siRNAs in the establishment of nucleolar dominance in A. suecica.