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A multidisciplinary study to investigate Herbaspirillum seropedicae-mediated priming in the wheat holobiont
No full textWheat crops represent a primary source of energy and nutrients for humans, providing staple food for an estimated 35% of the world population. However, the excessive use of synthetic fertilizers and pesticides has posed serious threaten to wheat production, calling for the need of more sustainable strategies to increase productivity while maintaining the ecological footprint as small as possible. In such circumstances, a great deal of help could be provided by the microbial world associated to plants.
All living plants are holobionts, consisting of a host plant and its associated microbiota. Microbiota includes diverse species of fungi and bacteria that can extend the plant´s ability to adapt to the changing environment, as they can provide nutrients and protect plants against biotic and abiotic stresses. Therefore, exploiting plant-associated microorganisms has gained substantial attention as a promising approach to create “greener” solutions to both increase crop productivity while maintaining the ecological footprint as small as possible and to minimize the use of synthetic fertilizers and pesticides. Inoculation of plants with beneficial plant growth-promoting bacteria (PGPB) causes both genetic and metabolic imprints in the host, leading to a pre-conditioned state known as priming. Priming causes changes in host metabolism, acting as a stress memory to provide plants with more sustained responses to future stresses at low energetic expenses. Furthermore, effects of PGPB inoculation have been shown to be also reflected by changes in the structure and dynamics of the plant-indigenous microbiota, causing transient structural shifts in microbiota diversity and often promoting plant-beneficial members. Although microbiota-modulation events have never been included in the definition of priming, shifts in plant-indigenous communities are believed to be crucial in determining the trajectories of plant growth and health. Herbaspirillum seropedicae is a PGPB isolated from various cereal plants which has the potential for an intimate root association and growth promotion in wheat. However, the mechanisms of action lying behind the growth-promoting effects of this bacterium remain unknown. In particular, there are no evidences regarding its role as driving force for the onset of a primed state both at the holobiont´s metabolome- and microbiota-level. In such framework, the present thesis investigated the priming effect induced by H. seropedicae from a holobiont perspective. First, a multidisciplinary approach combining culturomics, genetics and targeted metabolomics approaches was used to explore putative functional changes in the wheat bacteriota in response to H. seropedicae. This analysis showed that primed plants harbored a different bacterial community, which displayed a different capacity to modulate the levels of key Trp-related metabolites, supporting the idea a mode of action of this PGPB relying on a shift in both the composition and functionality of the wheat indigenous community. Changes in the structure of the endophytic wheat microbiota were then confirmed both by 16S and ITS metabarcoding analyses, and these shifts mirrored a predicted functional diversification, which was manifested by the enrichment of microbial members with potential beneficial functions for plant growth. Furthermore, untargeted metabolomic analyses were employed to explore the alterations in the root metabolome of primed plants. The obtained preliminary results showed that alterations of different metabolites with important roles in plant growth and defense, as well as in plant-microbe and microbe-microbe crosstalk. These metabolome shifts may be related with a preventive/alert state induced by priming, where specific precursor molecules acting at different metabolic steps of the same pathway may preferentially accumulate in the host as long-term memory.
In addition, the priming effect was also studied in wheat-pathogen interactions, where primed plants that were leaf-challenged with the pathogen Pseudomonas syringae showed both reduced oxidative stress and reduced necrotic symptoms. In this scenario, H. seropedicae may increase plant sensitivity to H2O2 signaling in systemic tissues, avoiding its massive accumulation upon pathogen challenge. These systemic effects may be linked not only to the plant oxidative metabolism itself, but also to the shifts in the leaf microbiota of primed plants, where specific members may be better adapted to counteract pathogen multiplication or buffer the disease-related cellular oxidative bursts.
The results obtained throughout the realization of this thesis suggest that priming with H. seropedicae not only has the potential to help plants to respond better to stress, but also to promote an invisible microbial machinery that is potentially beneficial for the host. The highly innovative and multidisciplinary approaches used throughout this thesis contributed to the disclosure of a novel mode of action of H. seropedicae to induce a primed state in the wheat holobiont. Understanding how native microbial communities interact with PGPB could yield important insights into how the plant microbiota can be exploited to create valuable and more eco-friendly solutions for agricultural application, such as biofertilizers and biopesticides
Utilização de amoxicilina/clavulanato na peritonite secundária com sépsis
No full textTrabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2024Introdução: Atualmente, está recomendada a utilização de antibioterapia empírica de largo espetro na peritonite secundária adquirida na comunidade de maior gravidade, porém estudos recentes apontam que uma antibioterapia empírica apropriada não está associada a um benefício claro em termos de sobrevivência pelo impacto do controlo de foco neste contexto. Assim, dada a importância da utilização racional dos antibióticos pelo problema crescente das resistências microbianas, este trabalho pretende avaliar a eficácia e segurança da utilização de antibioterapia de menor espetro, como a amoxicilina/clavulanato, nas peritonites secundárias de maior gravidade e quantificar as suas taxas de falência microbiológica. Métodos: Foi realizado um estudo retrospetivo com doentes submetidos a cirurgia gastrointestinal urgente, no contexto de peritonite secundária adquirida na comunidade e internados no Serviço de Medicina Intensiva (SMI) do Hospital de Santa Maria, entre janeiro de 2013 e abril de 2018. Colheram-se dados demográficos, clínicos, microbiológicos e de terapêutica. O efeito da administração de amoxicilina/clavulanato versus outro antibiótico foi avaliado quanto ao tempo de internamento e mortalidade hospitalar e no SMI, utilizando regressão linear e logística uni e multivariada. Resultados: Dos 216 doentes críticos incluídos, 51 realizaram antibioterapia empírica com amoxicilina/clavulanato. 94 doentes apresentaram isolamento bacteriano, sendo que 64.9% eram sensíveis a amoxicilina/clavulanato. A mortalidade no SMI nos doentes que realizaram amoxicilina/clavulanato e que realizaram outra antibioterapia empírica não apresentou uma diferença estatisticamente significativa (14.0% vs 24.0%, p = 0.11), tal como a mortalidade hospitalar (25.0% vs 40.0%, p = 0.07). Nos doentes que fizeram amoxicilina/clavulanato, o isolamento microbiológico não ser sensível a esta associação, não influenciou o tempo de internamento ou a mortalidade hospitalar e no SMI. Conclusões: A utilização de amoxicilina/clavulanato não teve um impacto negativo na mortalidade no SMI, mesmo quando se provou a posteriori como não tendo atividade in vitro contra os microrganismos isolados. Contudo, a taxa de falência microbiológica objetivada não permite utilizar esta antibioterapia com segurança na peritonite secundária de maior gravidade.Background: Currently, the use of empiric antimicrobial regimens with broad-spectrum activity is recommended in high-risk community-acquired secondary peritonitis. However, recent studies suggest that the appropriateness of empiric antimicrobial therapy is not clearly associated with survival benefit, due to the importance of source control in this context. Consequently, given the imperative of judicious antibiotic utilization due to the escalating problem of microbial resistance, this study aims to assess the efficacy and safety of using narrower spectrum antibiotics, such as amoxicillin/clavulanate, in severe secondary peritonitis and quantify the rates of microbiological failure associated with this approach. Methods: A retrospective study was conducted with patients undergoing urgent gastrointestinal surgery, in the context of community-acquired secondary peritonitis and admited to the ICU of Hospital de Santa Maria from January 2013 to April 2018. Demographic, clinical, microbiological, and therapeutic data were collected. The effect of amoxicillin/clavulanate administration versus other antibiotic was compared regarding the length of hospital stay and hospital and ICU mortality, using uni and multivariate linear and logistic regression analyses. Results: Among the 216 critically ill patients included in this study, 51 received amoxicillin/clavulanate. Bacterial isolation occurred in 94 patients, with 64.9% exhibiting sensitivity to amoxicillin/clavulanate. The ICU mortality rate among patients who underwent treatment with amoxicillin/clavulanate and those who received other empirical antibiotic therapy did not show a statistically significant difference (14.0% vs 24.0%, p = 0.11), nor did the hospital mortality rate (25.0% vs 40.0%, p = 0.07). Furthermore, microbiological isolation demonstrating resistance to amoxicillin/clavulanate did not significantly influence length of hospital stay or hospital and ICU mortality rates among patients who had received this therapy. Conclusions: The administration of amoxicillin/clavulanate did not adversely impact ICU mortality rate, even in cases where subsequent microbiological analysis revealed limited in vitro activity against the isolated microorganisms. However, the observed microbiological failure rate does not allow the use of this antibiotic therapy with safety in severe secondary peritonitis
A paz, o pão, ..., saúde, educação: a habitação, a grande ausência do Estado social democrático
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Seguimento do neurodesenvolvimento do recém-nascido grande prematuro em Portugal
No full textTrabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2024Até à idade do termo, a mortalidade e morbilidade nos recém-nascidos é, de uma forma global, inversamente proporcional à sua idade gestacional no momento do parto. A sobrevivência de recém-nascidos muito prematuros só é possível graças a técnicas avançadas de suporte disponíveis em algumas Unidades de Neonatologia, moAvando frequentemente internamentos prolongados. Além da ameaça à a sua sobrevivência, as crianças que ultrapassam esta fase têm riscos de saúde significaAvos, nomeadamente em relação ao neurodesenvolvimento. Consequentemente, surgiram por todo o mundo protocolos de seguimento, a fim de homogeneizar a avaliação dos recém-nascidos que se encontram em risco pela sua prematuridade. Estas recomendações, no entanto, variam de país para país, e a sua aplicação pode variar, inclusivamente, dentro de cada país, condicionada pelos recursos existentes. Este trabalho visa obter um panorama do seguimento dos recém-nascidos de idade gestacional inferior a 32 semanas que têm alta das Unidades de Neonatologia em Portugal, através do preenchimento de um quesAonário que explora diversos componentes desta avaliação longitudinal, tais como critérios de início e fim de seguimento, consultas previstas, profissionais envolvidos, instrumentos uAlizados e referenciação. A análise comparaAva dos diversos modelos poderá ser uma plataforma de lançamento para elaborar medidas de uniformização, à luz das melhores práAcas internacionais, a fim de obter um modelo exequível e consistente, que permita contribuir para o desenvolvimento de programas de seguimento mais completos, e para a avaliação comparaAva dos recém-nascidos de idade gestacional inferior a 32 semanas nascidos em Portugal.Up to term, mortality and morbidity in newborns is inversely proporAonal to their gestaAonal age at birth. The survival of very premature newborns is only possible thanks to advanced support techniques available in some Neonatology Units, o[en leading to prolonged hospital stays. In addiAon to the threat to their survival, children who go pass this stage present with significant health risks, parAcularly when it comes to neurodevelopment. As a result, follow-up protocols have emerged all over the world to standardize the assessment of newborns who are at risk due to their prematurity. These recommendaAons, however, vary from country to country, and their applicaAon can even vary within each country, condiAoned by exisAng resources. This study aims to obtain an overview of the follow-up of infants born prior to 32 completed weeks’ gestaAon discharged from Neonatology Units in Portugal, by compleAng a quesAonnaire that explores various components of this longitudinal assessment, such as criteria for the start and end of follow-up, planned appointments, professionals involved, instruments used and referrals. The comparaAve analysis of the various models could be a launching pad for drawing up standardizaAon measures, in the light of internaAonal best pracAces, to obtain a feasible and consistent model that can contribute to more complete follow-up programs, and to a comparaAve assessment of infants born prior to 32 weeks’ gestaAon delivered in Portugal
VIP neuroepithelial circuit in immunity and metabolism
No full textDissertação de Mestrado Integrado em Medicina Veterinária, área científica de Morfologia e FunçãoABSTRACT - In recent years, a multitude of studies have highlighted the coexistence of immune and neuronal cells in shared anatomical locations, where they synergistically interact to form neuroimmune cell units...RESUMO - Nos últimos anos, uma multiplicidade de estudos tem destacado a coexistência de células imunes e neuronais em locais anatómicos compartilhados, onde interagem sinergicamente para formar unidades celulares neuroimunes...N/
New synthetic strategies and structural optimization of the sugar moiety from a selective butyrylcholinesterase inhibitor
No full textThis PhD thesis focused on the synthesis and development of new synthetic strategies towards carbohydrate-based compounds with potential relevance for biological applications. Firstly, the development of a new synthetic methodology to prepare bicyclic sugar moieties bearing a fused furan ring was achieved. This strategy relies on a key Wittig reaction to afford the branched chain at position 3 of the sugar moiety with the required stereochemistry. The regio- and stereo-control was possible through optimization of the reaction conditions, as well as careful construction of the substrate. The presence of a bulky 2-O-pivaloyl group and the use of chloroform as solvent, led to the required (3E)-alkene by reaction of the 3-ulose with [(ethoxycarbonyl)methylene]triphenylphosphorane, while a 2-O-benzoyl protection afforded the corresponding (3Z)-alkene. When using acetonitrile as the reaction solvent, a (2Z)-alkene was formed regardless of the protecting group chosen. This domino reaction occurs through a keto-enol tautomerization, followed by an intramolecular migration of the acyl group at position 2 to position 3, and formation of the corresponding 2-ulose, which finally reacts with the phosphorane to afford the corresponding (2Z)-alkene. These results were significant towards the construction of bicyclic sugar moieties bearing a fused furan ring, present in several biological important compounds, such as the miharamycins sugar moiety as well as a selective butyrylcholinesterase inhibitor.
A library of purine nucleosides derived from a previously known selective butyrylcholinesterase inhibitor, the α-anomer of a fully benzylated sugar moiety N7 linked to a 2-acetamidopurine, was also investigated. Although this compound was a strong candidate for drug development, it presented a few pharmacokinetic problems, such as its water solubility and a high molecular weight, due to the high number of benzyl protection groups in the sugar moiety. With the goal of reducing the overall molecular weight, different patterns of protection of the sugar moiety were devised to access the significant positions for the displayed activity. Since an ether protection was necessary as previous studies showed no activity for ester protected sugar moieties, the smaller methyl group was chosen. A strategy was devised to regioselectively protect the different positions of the gluco sugar core by decreasing the number of benzyl groups present in the final compound, until a per-methylated glycosyl donor was synthesized. The presence of a benzyl group at positions 2 and 3 led to a slight decrease of the βN7 yield when compared to the fully benzylated purine nucleoside. When these positions were protected with a methyl group, selectivity towards the βN9 isomer was observed as no αN7 was detected and the βN7 yield was significantly decreased from ≈ 25 % to ≈ 9 %. Biological assays from the synthesized nucleosides, with both acetylcholinesterase and butyrylcholinesterase, showed a significant decrease in the activity of these compounds when compared to the fully benzylated derivative, and the most active compound was 2,3-di-O-benzyl-4,6-di-O-methyl αN7 purine nucleoside, although it showed no selectivity towards butyrylcholinesterase (Kia = 12 μM for AChE, Kia = 83 μM for BuChE). As displayed by these and the previous results, the αN7 isomer seems to favor activity towards cholinesterases. Although this isomer gives the best results, the reaction conditions did not favor the formation of this compound as it was obtained in yields lower than 10 %. The next step was to investigate reaction conditions to improve the αN7 yield. For this, changing the starting material with different leaving groups at the anomeric position as well as different Lewis acid activation in the N-glycosylation were tested. These changes did not present significant improvements, so the reaction was tested at different temperatures, where an increase in the αN7 yield was observed when the reaction was carried out at room temperature when compared to the previously reported temperature of 65 °C.
A new analogue of the lead compound was synthesized to perform biodistribution in vivo studies. A 6-bromo purine nucleoside was obtained to undergo a SN2 reaction with a radioiodinated reagent to afford a radioactive labelled compound. This compound was then injected into healthy CD1 mice and results were measured at 2- and 60- min post-injection. The low brain uptake (0.36 % and 0.10 % at 2- and 60- min respectively) suggests that only a low fraction of the radioiodinated compound crosses the BBB. These results encouraged the docking studies of the active site of BuChE with the lead compound. Key interactions were identified with the acetamido group of the purine and the benzyl group from position 2. Since different protecting groups showed lower activity, deoxygenation was attempted at selected positions of the sugar moiety. A regioselective synthetic strategy was then developed towards 4 different deoxygenated sugar moieties. Results showed that removing the benzyloxy group at position 6 or using the corresponding lyxo-pentopyranoside still maintained selective inhibition of butyrylcholinesterase. The most active compounds were also tested for their cytotoxicity and showed low cytotoxicity at the active concentrations
Baseline description of inorganic contaminants in juvenile sharks off the Boa Vista island nursery area (Cabo Verde)
No full textTese de mestrado, Ecologia Marinha , 2024, Universidade de Lisboa, Faculdade de CiênciasUnderstanding the health of local shark populations is crucial to assessing the impacts of human
activity on marine ecosystems and promoting the sustainable use of their resources. This study
establishes the first baseline data on the levels of inorganic contaminants (Al, Zn, As, Cu, Cr, Cd, Co,
Mn, Ti, Ni, Hg, and Pb) in juvenile sharks from the nursery area of Sal Rei (Boa Vista island, Cabo
Verde), focusing on four species: milk shark (Rhizoprionodon acutus), scalloped hammerhead shark
(Sphyrna lewini), blacktip shark (Carcharhinus limbatus), and Atlantic weasel shark (Paragaleus
pectoralis). Plasma samples from these sharks, along with sediment and sea water samples, were
collected from the study area. Subsequently, the elemental concentrations of all samples were
determined using total reflection X-ray fluorescence spectroscopy (TXRF S2 PICOFOX, Brucker,
Germany). For a better characterization of the sediments, we also performed their granulometric
description and applied pollution indices: Enrichment Factor (EF), Risk Index (RI), and Metal Pollution
Index (MPI). Only the MPI was performed to the sea water samples.
The elemental analyses of the abiotic factors revealed a generally low contaminated
environment. However, a slight anthropogenic elemental increase was detected through the pollution
indices in specific sampling locations, particularly in sites 1 and 2. These sites are near the fishing port
of Sal Rei, and additional contamination is likely due to the fishing activities of local residents.
As expected, the sharks exhibited low levels of inorganic contaminants, consistent with the
relatively undisturbed environment. However, interspecific differences were observed, with blacktip
sharks showing higher concentrations of copper (Cu) and milk and Atlantic weasel sharks showing
significantly higher arsenic (As) levels compared to the other species. Principal Component Analysis
(PCA) revealed distinct elemental signatures between the species, particularly between blacktip sharks
and Atlantic weasel sharks, likely due to differences in their respective feeding ecologies
Three Decades of Research on Iberian Wild Ungulates: Key Insights and Promising Research Avenues
No full textThe Iberian Peninsula is witnessing ever-faster environmental changes, and new challenges for wild ungulates are continuously emerging as they become more abundant and widespread.
We conducted a systematic review to analyse the knowledge on wild ungulates inhabiting the Iberian Peninsula. We used Web of Science and Scopus search engines, complemented by searches in nonindexed journals, to examine peer-reviewed articles published between January 1990 and July 2023.
The annual average growth rate in the number of publications was 17%. Most studies focussed on diseases and pathogens (36%), physiology, endocrinology and reproduction (19%), and behaviour, population and community ecology (17%). Red deer and wild boar are the most targeted species in scientific literature, followed by the Iberian wild goat, roe deer, fallow deer, Southern chamois, mouflon and aoudad.
We identify key knowledge gaps that deserve further attention such as the ecological and social impacts of (re)introductions, the effects of increasing ungulate densities on ecosystem integrity and the impact of different hunting and management techniques (some unique to the Iberian Peninsula) on population dynamics. We also highlight the need to stimulate Iberian collaboration and extend the discussion to a wider range of stakeholders to integrate different perspectives on the research agenda for Iberian wild ungulates.info:eu-repo/semantics/acceptedVersio
Grapevine subtilisin-like proteases and Plasmopara viticola serine protease inhibitors in the defence – offense interaction
No full textGrapevine (Vitis vinifera L. subsp. sativa) is one of the most cultivated fruit crops worldwide. However, is highly susceptible to several pathogens which poses a severe threat to production yields. One of the most devastating diseases is the grapevine downy mildew caused by the obligate biotrophic oomycete Plasmopara viticola (Berk. & M.A. Curtis) Berl. & De Toni. This pathogen was introduced in Europe in the mid19th century and led to the near-collapse of European viticulture only some years later. To manage this disease several preventive applications of pesticides are used but more sustainable approaches such as breeding for resistance are being conducted. As such, most of the research in the field of grapevine-pathogen interaction is still focused on increasing knowledge of how the plant activates defence pathways. However, the success of the breeding programs depends also on a deep knowledge of this pathosystem. Not only on the identification of strong resistance-associated candidate genes for introgression from the resistance carriers and hybrids, but as well as on the interacting players in the plant-pathogen offense-defence, as plant defence proteins/ enzymes and pathogen effectors. The activation of the defence pathways and therefore the establishment of an incompatible interaction depends on the early recognition of pathogens. Among the groups of molecules, that play a role in pathogen recognition and signalling cascades, we focused on plant proteases, particularly in serine proteases, or the subtilisin-like proteases (SBT, subtilases) family. In grapevine, some subtilases were observed to have an enhanced gene expression upon infection with P. viticola in the tolerant cultivar “Regent” and susceptible cultivar “Trincadeira”, as VviSBT4.19. Moreover, some subtilases, as VviSBT5.3, were found to have their expression induced not only in response to the pathogen but also by jasmonic acid, a phytohormone involved in the signalling pathway in biotic stress response. Focusing on the pathogen, apoplastic effectors such as protease inhibitors are necessary for a successful invasion and counter defence. In this context, one hypothesis is that P. viticola may secrete protease inhibitors to block host serine proteases. This doctoral project’s main aim is to understand the role of subtilases in grapevine resistance and the role of serine-protease inhibitors secreted by P. viticola as pathogenicity effectors, particularly to understand the potential crosstalk between grapevine and P. viticola focusing on host subtilases and pathogen serine proteases inhibitors. Several objectives were defined for the analysis of host serine proteases as the analysis of VviSBT4.19 and VviSBT5.3 gene expression in V. vinifera genotypes with different tolerance and Rpv background in a time-course inoculation with P. viticola. Stable transformation of Solanum lycopersicum with constructs for overexpression of VviSBT4.19 and SlSBT4.14, a tomato homolog of VviSBT4.19 to access gene function, and characterization of these Solanum lycopersicum transformed lines. For the analysis of serine protease inhibitors, we identified P. viticola protease inhibitors from the serine family, performed gene expression analysis of these inhibitors and analysis of their subcellular localization and their effect on plant defence mechanism by agroinfiltration in N. benthamiana. Gene expression analysis of two candidate subtilases, VviSBT4.19 and VviSBT5.3a, was performed in seven cultivars with different levels of susceptibility to P. viticola (Chapter II). Two Vitis vinifera susceptible genotypes: “Chardonnay” and “MuellerThurgau” and five tolerant genotypes “Calardis blanc” (Rpv3–1, Rpv3–2), “Cabernet blanc” (Rpv3–1), “Regent” (Rpv3–1), “Solaris” (Rpv10, Rpv3-3) and “Sauvignac” (Rpv12, Rpv 3 − 1) were analysed. Moreover, two P. viticola isolates with different degrees of virulence were tested, avrRpv3 + which is an isolate unable to overcome Rpv3-based resistance and NW-10/16 which was isolated from ‘Regent’ in October 2016 in Neustadt/Weinstr. (Germany). Not only NW-10/16 can overcome the resistance observed in Rpv3 genotypes when infected with the avirulent isolate avrRpv3+, in the susceptible cultivars, “Chardonnay” NW-10/16 also exhibits an increase in sporulation. This higher sporulation on susceptible cultivars might indicate that the isolate NW-10/16 presents a higher aggressiveness. The subtilases display different patterns of expression with the VviSBT4.19 exhibiting only one peak of expression, while VviSBT5.3a presents a bimodal behaviour. Nevertheless, both subtilases presented a more pronounced response to the more aggressive P. viticola isolate, with the difference in gene expression being also dependent on the grapevine cultivar. Our results support the hypothesis that the two subtilases are involved in the early events of the defence mechanisms, being their transcription highly activated in response to a more aggressive P. viticola isolate. The P. viticola isolates were further characterized in Chapter III. The interaction of these isolates with 5 grapevine genotypes with different Rpv loci was analysed. In this chapter, P. viticola development, aggressiveness and the expression of serine protease inhibitors was assessed. The aim was to distinguish two P. viticola isolates through their infection strategy and growth stages, and possible determine if a different gene expression of serine protease inhibitors could be a cause of one of the causes of the increased aggressiveness. The results showed that the isolate NW-10/16 was able to overcome Rpv3 resistance but not Rpv12 resistance. Moreover, it can be considered more aggressive with increased sporulation and increased hyphae growth, with an acceleration in hyphae growth after 36 hours post inoculation. P. viticola serine protease inhibitors gene family was characterized with eight genes encoding for five proteins. From these five serine protease inhibitors, two present highly dynamic pattern of expression depending on the isolate and cultivar. The Pvit020s, a serine protease inhibitor gene that has three identical copies in the genome, plus one containing a mutation which alters 1 aminoacid, present a later peak of expression in the susceptible cultivar but earlier in tolerant cultivars. While Pvit026 has an both a higher expression level and an earlier peak of expression for NW-10/16 compared to avrRpv3+. When comparing the gene expression of both isolates, NW-10/16 presented a higher expression for Pvit026 in all cultivars and Pvit544 in the tolerant and resistant cultivars. The expression patterns indicate a role of serine proteases inhibitors in the pathogen’s counterattack against plant defence. Moreover, we can hypothesize that Pvit026 may be involved in increased aggressiveness, while Pvit544 might have a role as a facilitator to overcome resistance. The function of subtilisin-like serine protease VviSBT4.19 was studied in Chapter IV with the stable transformation of Solanum lycopersicum to obtain transgenic plant lines overexpressing the grapevine VviSBT4.19. To compare, the homolog subtilisinlike serine protease gene from tomato, SlSBT4.14 was also cloned and used for stable transformation. Cotyledons of S. lycopersicum “Hellfrucht” were co-incubated with A. tumefaciens AGL1 for stable transformation. No difference in calli morphology was observed between constructs (T-DNA containing VviSBT4.19, SlSBT4.14 or none). However, calli transformed with the transgene VviSBT4.19 presented a slightly faster regeneration. Three generations of transgenic plant lines were analyzed. For each construct two transgenic plant lines were studied. Morphologically, the overexpression of VviSBT4.19 altered the angle of the tomato leaves, which tended to grow with a vertical orientation. And plants overexpressing SlSBT4.14 tended to be shorter. Another major difference was found in flowering and fruit development. Plants overexpressing VviSBT4.19 and SlSBT4.14 tended to flower later with some not producing fruits and seeds. The resistance to Phytophthora infestans was evaluated for the transgenic plant lines and plants overexpressing SlSBT4.14 were found to be more susceptible. And plants overexpressing VviSBT4.19 tend to present a lower pathogen sporulation, and therefore tend to be less susceptible to P. infestans infection. These results indicate that both subtilases have possible functions in biotic stress response as well as plant development and reproduction. Moreover, the overexpression of VviSBT4.19 and SlSBT4.14 resulted in very different phenotypes regarding growth and biotic stress response, further indicating that these subtilases may participate in different mechanisms. Though the exact mechanism of action remains uncertain. As it was possible to determine in which plant processes these subtilases have probable functions, new experiments can be design and carried out more specifically, and thus further elucidating the subtilases roles in these processes. This thesis is divided in four chapters. The first chapter was written as a review article and the 3 following chapters were written as scientific articles and each has its own abstract, introduction, materials and methods, results and discussion, conclusion, acknowledgments and references
Silêncios sociologicamente pertinentes: jovens adolescentes que não falam com ninguém sobre assuntos relacionados com sexualidade
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