Abordagens moleculares no estudo da diversidade microbiana

A importância dos microrganismos na manutenção da vida na Terra é reconhecida por todos. Estes organismos microscópicos são relevantes em áreas tão diversas como a Saúde, Agricultura, Indústria ou Ambiente. O número estimado de todas as espécies é de 100 milhões, das quais 2 milhões foram formalmente descritas. Das cerca de 1,5 a 3,5 milhões de espécies fúngicas que se estimam existir, apenas 5% se encontram descritas. Tradicionalmente, a identificação de espécies de fungos baseia-se no seu isolamento e análise da morfologia das suas estruturas reprodutoras. Embora de grande utilidade, a caracterização morfológica é limitada devido ao reduzido número de caracteres que podem ser analisados, da dependência das condições de cultura e das variações intrínsecas do microrganismo. Actualmente, métodos moleculares baseados na sequenciação do espaçador interno do transcrito de rDNA (ITS) têm vindo a ser utilizados na identificação eficaz de espécies fúngicas. Estudos populacionais têm recorrido à amplificação da região ITS e comparação dos perfis electroforéticos em gradientes desnaturantes (DGGE ou TGGE) de diferentes amostras. Recentemente, o desenvolvimento de técnicas de sequenciação massiva permitiram a emergência de uma nova área- a metagenómica - que pode ser aplicada a comunidades de microrganismos sem a necessidade de os isolar ou cultivar. Este facto adquire especial relevância por se estimar que apenas 17% dos fungos serão capazes de ser cultivados in vitro. Nesta apresentação serão abordados as estratégias que permitiram a evolução da metagenómica. Os desafios da identificação de microrganismos em comunidades serão discutidos, focando o caso particular da diversidade fúngica em amostras de solos.Financiado pela FCT, projecto PTDC/AGR-AAM/099556/200

Vantagens e desvantagens da relação entre plantas e fungos

Apresentação efetuada nas III Jornadas Micológicas, Braga, 2011Os fungos são capazes de estabelecer múltiplas associações/relações com as plantas. Apesar de muitas dessas relações se revelarem prejudiciais, outras apresentam vantagens significativas ao nível do desenvolvimento, nutrição e sanidade das plantas. Nesta comunicação será particularmente abordada a associação sirnbiótica que ocorre entre raízes e fungos do solo - micorrização - referindo-se as vantagens que daí advêm para plantas, fungos e inclusivamente para o Homem

Distinguishing allies from enemies-a way for a new green revolution

Plants are continually interacting in different ways and levels with microbes, resulting in direct or indirect effects on plant development and fitness. Many plant-microbe interactions are beneficial and promote plant growth and development, while others have harmful effects and cause plant diseases. Given the permanent and simultaneous contact with beneficial and harmful microbes, plants should avoid being infected by pathogens while promoting mutualistic relationships. The way plants perceive multiple microbes and trigger plant responses suggests a common origin of both types of interaction. Despite the recent advances in this topic, the exploitation of mutualistic relations has still not been fully achieved. The holistic view of different agroecosystem factors, including biotic and abiotic aspects, as well as agricultural practices, must also be considered. This approach could pave the way for a new green revolution that will allow providing food to a growing human population in the context of threat such as that resulting from climate change.This work was supported by FCT (Fundacao para a Ciencia e a Tecnologia) within the framework of CBMA (UIDB/04050/2020) and CIMO (UID/AGR/00690/2020) I&D centers

Role of label-retaining cells in estrogen-induced endometrial regeneration

Candidate stem/progenitor cells have been identified in mouse endometrium as label-retaining cells (LRCs). The role of endometrial stem/progenitor cells in initiating estrogen-stimulated endometrial growth in prepubertal and cycling mice was investigated following a single 17beta-estradiol (E2) injection in bromodeoxyuridine (BrdU)-labeled and -chased (LRC), ovariectomised mice. Proliferating (BrdU(+)/Ki-67(+)) and mitotic (BrdU(+)/PH3(+)) epithelial LRCs were first detected in prepubertal mice 8 hours following E2 treatment, initiating the proliferative response. In contrast, all epithelial LRCs and 16% of epithelial cells in cycling mice proliferated within 2 hours. In cycling mice, 12% of stromal LRCs initiated a proliferative response 8 hours after E2. Proliferating epithelial LRCs and most stromal LRCs (85%) lacked estrogen receptor-alpha (ESR1). These findings suggest that endometrial epithelial LRCs function as stem/progenitor cells by receiving proliferative signals from neighboring ESR1(+) niche cells to initiate the growth of the epithelium during development, while mature epithelial cells may undergo self-replication in cycling endometrium.postprin

An improved method for high-quality RNA isolation from needles of adult maritime pine trees

Conventional RNA isolation methods optimised for pine seedlings have been shown to produce poor quality RNA when applied to needles of adult pine trees. We describe here a modified procedure to isolate high pure RNA from needles of thirty-year-old maritime pines, exhibiting high levels of phenolics, polysaccharides and RNases. Major changes were the inclusion of proteinase K in the extraction medium followed by incubation at 42°C. Integrity and purity were evaluated by denaturing gel electrophoresis and by spectrophotometry (A260/A230 and A260/A280). Total RNA could be successfully used for poly(A)+-RNA isolation and cDNA library construction.Fundação para a Ciência e a Tecnologia (FCT) - grant ref. SFRH/BD/3194/2000

Molecular aproach on the study of infection of Pinus pinaster Ait. by the fungus Lophodermium seditiosum

Poster apresentado no 10th International Congress on Molecular Plant-Microbe Interactions, Madison, Wisconsin, Estados Unidos da América.The fungi genus Lophodermium comprises several species known to intervene in the degradation process of pine needles. Amongst these species, only L. seditiosum is known to be pathogenic, as it is able to infect healthy needles in young pine populations, spreading from stomata and leading to the death of the needle (needle cast disease). Maritime pine (Pinus pinaster, Ait) is one of the most relevant forest species in SW Europe. Needle cast disease, by attacking young plants, leads to two main events: the lack of self regeneration in natural populations, and complete destruction of pine nurseries. The purpose of our study is to understand the nature of the infection of Pinus pinaster Ait. by L. seditiosum and ultimately gain some knowledge on the overall response of Conipherophyta to infection. As a first step towards this goal, a cDNA library of Pinus pinaster was constructed. Subsequent effort has been directed towards the screening of genes encoding PR proteins involved in host-pathogen interaction, as well as oxidative stress enzymes. In addition, PCR amplification using degenerate primers is in progress in order to allow the identification of putative Resistance genes.Fundação para a Ciência e a Tecnologia (FCT) - grant ref. SFRH/BD/3194/200

Phytochemicals properties and antioxidant activities of some accessions of Phaseolus vulgaris L

Common bean is a legume important in Mediterranean diet with interesting nutritional food characteristics due to its proteins, fibers and antioxidant activity. There is an increased interest among consumers in foods rich in natural antioxidants for maintaining and improving health. Beyond this, beans are a low-price food and an excellent complement for the population with low income. There are some differences in the phenolic and flavonoid compounds content and antioxidant capacity between accessions of common bean. In this study, 15 traditional Portuguese and 1 Greek accessions of Phaseolus vulgaris L. were analyzed. Total phenols, ortho-diphenols and flavonoid contents were investigated and antioxidant activity were measured by different assays (DPPH, ABTS) which may give a better characterization of this activity

Magnetophoresis of Nonmagnetic, Submicrometer Particles in Magnetic Fluids

We studied the migration of nonmagnetic, submicrometer polystyrene beads submerged in a magnetic fluid in the presence of nonuniform magnetic fields as a potential method for size-based separation of submicrometer, nonmagnetic species. Since the polystyrene beads are much larger than the magnetic fluid nanoparticles, the magnetic fluid was treated as a one-component continuum with respect to the beads. We found that the polystyrene beads will migrate in the direction of decreasing magnetic fields and will focus over a region where the magnetic field or its gradient vanishes, as predicted by our model. The concentration profiles predicted by our model, which has no adjustable or fitted parameters, agree reasonably well with the experimental data both qualitatively and quantitatively.Singapore-MIT Alliance (SMA

Transcriptome of Quercus suber challenged by drought, salinity, and oxidative stresses

Comunicação em painelThe cork oak (Quercus suber) forest (the “montado”) is a unique and emblematic resource for Portugal, due to its ecological, socio-economic significance, and the commercial value of cork. Plant abiotic stresses, particularly reduced water availability and extreme temperatures, are substantial constraints to agricultural and agro-forestry production. In the particular case of cork oak, the damaging land-use policies, the climate change, and imposition of abiotic stresses related with high light intensities have been threatening the cork oak forests. The adaptation to abiotic stresses comprises a series of morphological, physiological, biochemical and molecular changes controlled by complex molecular networks. Recent advances in increasing plant tolerance were achieved after the identification of specific genes suited for plant genetic engineering. However, as abiotic stress is commonly present in the field as a combination of different stresses, the complex plant response mechanisms are far from being elucidated. This work is part of a coordinated effort to uncover the transcriptome of Q. suber and attention was paid to the identification of expressed sequence tags (ESTs) involved in responses to distinct abiotic stress challenges, namely drought, salt and oxidative stresses.This work was supported by the FCT project SOBREIRO/0033/200

Treatment of tumour recurrence after resection of hepatocellular carcinoma. Analysis of 97 consecutive patients

OBJECTIVE: To evaluate the long-term results of aggressive treatment of HCC recurrence. METHODS: Two hundred and nine consecutive patients underwent hepatic resection for HCC in our hospital. Tumour recurrence was diagnosed in 97 (51%) of the 190 patients with curative resection. Sixteen underwent hepatic resection: two right hepatectomies, one three-segmentectomy, one left hepatectomy, five two-segmentectomies, six segmental resections and one subsegmentectomy. Two patients with metastasis in the spine were submitted to a vertebral body resection. Twenty-five patients were treated with percutaneous ethanol injection or intra-arterial chemoembolization. Fifty-four patients with a poor performance status and liver function or multiple extra hepatic recurrences did not receive any treatment. RESULTS: There were no operative deaths. The postoperative mortality rate was 5.5% (one patient). The cumulative overall survival after the second resection was respectively 89%, 46% and 31% at 1, 3 and 5 years. There was a significant difference in survival between patients treated with repeat resection and those submitted to a non-surgical or conservative treatment (p<0.0001). There were no differences in operative deaths, postoperative mortality and morbidity between the first and second hepatic resection. CONCLUSIONS: Aggressive management with combined resection or loco regional therapy for intrahepatic recurrence and resection of isolated extra-hepatic recurrence may offer long-term survival in selected patients. Second liver resection for recurrence of HCC can be safely performed