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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.