Genetic diversity of stay-green sorghums and their derivatives revealed by microsatellites

The genetic variability of 28 sorghum genotypes of known senescence phenotype was investigated using 66 SSR markers well-distributed across the sorghum genome. The genotypes of a number of lines from breeding programmes for stay-green were also determined. This included lines selected phenotypically for stay-green and also RSG 03123, a marker-assisted backcross progeny of R16 (recurrent parent) and B35 (stay-green donor). A total of 419 alleles were detected with a mean of 6.2 per locus. The number of alleles ranged from one for Xtxp94 to 14 for Xtxp88. Chromosome SBI-10 had the highest mean number of alleles (8.33), while SBI-05 had the lowest (4.17). The PIC values obtained ranged from zero to 0.89 in Xtxp94 and Xtxp88, respectively, with a mean of 0.68. On a chromosome basis, mean PIC values were highest in SBI-10 (0.81) and lowest in SBI-05 (0.53). Most of the alleles from B35 in RSG 03123 were found on chromosomes SBI-01, SBI-02 and SBI-03, confirming the successful introgression of quantitative trait loci associated with stay-green from B35 into the senescent background R16. However, the alternative stay-green genetic sources were found to be distinct based on either all the SSRs employed or using only those associated with the stay-green trait in B35. Therefore, the physiological and biochemical basis of each stay-green source should be evaluated in order to enhance the understanding of the functioning of the trait in the various backgrounds. These genetic sources of stay-green could provide a valuable resource for improving this trait in sorghum breeding programmes.

Effects of organic fertilizers on the vegetative, nutritional, and productive parameters of blueberries ‘Corona’, ‘Legacy’, and ‘Liberty’

Organic farming does not allow using certain inputs, such as N, which differ in nutrient release rates and dynamics. To evaluate the effect of different organic fertilizers on the vegetative, nutritional, and productive parameters of blueberries ( Vaccinium corymbosum L.), a pot experiment was conducted in three consecutive seasons in a sandy soil of south-central Chile using ‘Corona’, ‘Legacy’ and ‘Liberty’. The following fertilizers were evaluated: compost (CM), Purely Grow (PG), Purely Lysine (PL), Fertil (F), blood meal (BM), lupine meal (LM), along with a control treatment without fertilization (C) and two conventional treatments with urea (CF) and sodium nitrate (S). Results indicate that vegetative growth and leaf N concentration prior to senescence were different among cultivars in the three evaluated seasons. The highest leaf N concentration was recorded in ‘Corona’ followed by ‘Legacy’ and ‘Liberty’ while levels tended to increase in the seasons. Quick-release N sources had greater effects on these parameters but with differences among cultivars. Fruit yield and weight were higher in ‘Corona’ followed by ‘Legacy’ and ‘Liberty’. Fruit yield was generally higher when using LM and F and showed no effect on fruit weight. Leaf chlorophyll content was higher in ‘Corona’ followed by ‘Legacy’ and ‘Liberty’, which increased when using CF, LM, BM, and PG. Finally, the organic fertilizer and blueberry cultivar that obtained the highest values for most of the evaluated parameters were LM and Corona, respectively.

From the field to the table: ionizing radiation as a feasible postharvest treatment for fresh and dried plant foods

Food irradiation is a treatment that involves subjecting in-bulk or packaged food to a controlled dose of ionizing radiation, with a clearly defined goal. It has been used for disinfestation and sanitization of food commodities and to retard postharvest ripening and senescence processes, being a sustainable alternative to chemical agents 1 . Doses up to 10 kGy are approved by several international authorities for not offering negative effects to food from a nutrition and toxicology point of view 2 . However, the adoption of this technology for food applications has been a slow process due to some misunderstandings by the consumer who often chooses non-irradiated foods. In this study, the effects of the ionizing radiation treatment on physical, chemical and bioactive properties of dried herbs and its suitability for preserving quality attributes of fresh vegetables during cold storage were evaluated. The studied herbs, perennial spotted rockrose (Tuberaria lignosa (Sweet) Samp.) and common mallow (Malva neglecta Wallr.) were freeze-dried and then irradiated up to 10 kGy in a Cobalt-60 chamber. The selected vegetables, watercress (Nasturtium officinale R. Br.) and buckler sorrel (Rumex induratus Boiss. Reut.) were rinsed in tap water, packaged in polyethylene bags, submitted to irradiation doses up to 6 kGy and then were stored at 4 C for a period of up to 12 days. Physical, chemical and bioactive parameters of irradiated and non-irradiated samples were evaluated using different methodologies the colour was measured with a colorimeter, individual chemical compounds were analyzed by chromatographic techniques, antioxidant properties were evaluated using in vitro assays based on different reaction mechanisms, and other quality analyses were performed following official methods of analysis. The irradiation treatment did not significantly affect the colour of the perennial spotted rockrose samples, or its phenolic composition and antioxidant activity 3 . Medium doses preserved the colour of common mallow and a low dose did not induce any adverse effect in the organic acids profile. The green colour of the irradiated vegetables was maintained during cold storage but the treatment had pros and cons in other quality attributes. The 2 kGy dose preserved free sugars and favoured polyunsaturated fatty acids (PUFA) while the 5 kGy dose favoured tocopherols and preserved the antioxidant properties in watercress samples. The 6 kGy dose was a suitable option for preserving PUFA and the ω-6 ω-3 fatty acids ratio in buckler sorrel samples. This comprehensive experimental work allowed selecting appropriate processing doses for the studied plant foods in order to preserve its quality attributes and edibility.

Bcl-xL (S49) and (S62) sequential phosphorylation/dephosphorylation during mitosis prevents chromosome instability and aneuploidy

Une caractéristique intéressante de la protéine Bcl-xL est la présence d'un domaine en boucle non-structurée entre les hélices α1 and α2 de la protéine. Ce domaine protéique n'est pas essentiel pour sa fonction anti-apoptotique et absent chez CED-9, la protéine orthologue chez Caenorhabditis elegans. A l'intérieur de ce domaine, Bcl-xL subit une phosphorylation et déphosphorylation dynamique sur les résidus Ser49 et Ser62 en phase G2 du cycle cellulaire et lors de la mitose. Lorsque ces résidus sont mutés et les protéines exprimées dans des cellules cancéreuses, les cellules démontrent plusieurs défauts mitotiques liés à l'instabilité chromosomique. Pour analyser les effets de Bcl-xL Ser49 et Ser62 dans les cellules normales, les présentes études ont été réalisées dans des cellules diploïdes humaines normales, et in vivo chez Caenorhabditis elegans. Dans une première étude, nous avons utilisé la lignée cellulaire de cellules fibroblastiques diploïdes humaines normales BJ, exprimant Bcl-xL (type sauvage), (S49A), (S49D), (S62A), (S62D) et les double (S49/62A) et (S49/62D) mutants. Les cellules exprimant les mutants de phosphorylation ont montré des cinétiques de doublement de la population cellulaire réduites. Ces effets sur la cinétique de doublement de la population cellulaire corrèle avec l'apparition de la sénescence cellulaire, sans impact sur les taux de mort cellulaire. Ces cellules sénescentes affichent des phénotypes typiques de sénescence associés notamment à haut niveau de l'activité β-galactosidase associée à la sénescence, la sécrétion d' interleukine-6, l'activation de p53 et de p21WAF1/ Cip1, un inhibiteur des complexes kinase cycline-dépendant, ainsi que la formation de foyers de chromatine nucléaire associés à γH2A.X. Les analyses de fluorescence par hybridation in situ et des caryotypes par coloration au Giemsa ont révélé que l'expression des mutants de phosphorylation de Bcl-xL provoquent de l'instabilité chromosomique et l'aneuploïdie. Ces résultats suggèrent que les cycles de phosphorylation et déphosphorylation dynamiques de Bcl-xL Ser49 et Ser62 sont importants dans le maintien de l'intégrité des chromosomes lors de la mitose dans les cellules normales. Dans une deuxième étude, nous avons entrepris des expériences chez Caenorhabditis elegans pour comprendre l'importance des résidus Ser49 et Ser62 de Bcl-xL in vivo. Les vers transgéniques portant les mutations de Bcl-xL (S49A, S62A, S49D, S62D et S49/62A) ont été générés et leurs effets ont été analysés sur les cellules germinales des jeunes vers adultes. Les vers portant les mutations de Bcl-xL ont montré une diminution de ponte et d'éclosion des oeufs, des variations de la longueur de leurs régions mitotiques et des zones de transition, des anomalies chromosomiques à leur stade de diplotène, et une augmentation de l'apoptose des cellules germinales. Certaines de ces souches transgéniques, en particulier les variants Ser/Ala, ont également montré des variations de durée de vie par rapport aux vers témoins. Ces observations in vivo ont confirmé l'importance de Ser49 et Ser62 à l'intérieur du domaine à boucle de Bcl-xL pour le maintien de la stabilité chromosomique. Ces études auront une incidence sur les futures stratégies visant à développer et à identifier des composés qui pourraient cibler non seulement le domaine anti-apoptotique de la protéine Bcl-xL, mais aussi son domaine mitotique pour la thérapie du cancer.

Transcriptoma diferencial entre células-tronco mesenquimais humanas jovens e senescentes

Human mesenchymal stem cells (MSC) are powerful sources for cell therapy in regenerative medicine. The long time cultivation can result in replicative senescence or can be related to the emergence of chromosomal alterations responsible for the acquisition of tumorigenesis features in vitro. In this study, for the first time, the expression profile of MSC with a paracentric chromosomal inversion (MSC/inv) was compared to normal karyotype (MSC/n) in early and late passages. Furthermore, we compared the transcriptome of each MSC in early passages with late passages. MSC used in this study were obtained from the umbilical vein of three donors, two MSC/n and one MSC/inv. After their cryopreservation, they have been expanded in vitro until reached senescence. Total RNA was extracted using the RNeasy mini kit (Qiagen) and marked with the GeneChip ® 3 IVT Express Kit (Affymetrix Inc.). Subsequently, the fragmented aRNA was hybridized on the microarranjo Affymetrix Human Genome U133 Plus 2.0 arrays (Affymetrix Inc.). The statistical analysis of differential gene expression was performed between groups MSC by the Partek Genomic Suite software, version 6.4 (Partek Inc.). Was considered statistically significant differences in expression to p-value Bonferroni correction ˂.01. Only signals with fold change ˃ 3.0 were included in the list of differentially expressed. Differences in gene expression data obtained from microarrays were confirmed by Real Time RT-PCR. For the interpretation of biological expression data were used: IPA (Ingenuity Systems) for analysis enrichment functions, the STRING 9.0 for construction of network interactions; Cytoscape 2.8 to the network visualization and analysis bottlenecks with the aid of the GraphPad Prism 5.0 software. BiNGO Cytoscape pluggin was used to access overrepresentation of Gene Ontology categories in Biological Networks. The comparison between senescent and young at each group of MSC has shown that there is a difference in the expression parttern, being higher in the senescent MSC/inv group. The results also showed difference in expression profiles between the MSC/inv versus MSC/n, being greater when they are senescent. New networks were identified for genes related to the response of two of MSC over cultivation time. Were also identified genes that can coordinate functional categories over represented at networks, such as CXCL12, SFRP1, xvi EGF, SPP1, MMP1 e THBS1. The biological interpretation of these data suggests that the population of MSC/inv has different constitutional characteristics, related to their potential for differentiation, proliferation and response to stimuli, responsible for a distinct process of replicative senescence in MSC/inv compared to MSC/n. The genes identified in this study are candidates for biomarkers of cellular senescence in MSC, but their functional relevance in this process should be evaluated in additional in vitro and/or in vivo assays

Étude fonctionnelle d’un nouveau complexe multi-enzymatique régulant l’épigénome

L’ubiquitination, une modification post-traductionnelle importante pour le contrôle de nombreux processus cellulaires, est une réaction réversible. La réaction inverse, nommée déubiquitination est catalysée par les déubiquitinases (DUB). Nous nous sommes intéressés dans nos travaux à étudier l’ubiquitination de l’histone H2A (H2Aub), au niveau des résidus lysines 118 et 119 (K118/K119), une marque épigénétique impliquée dans la régulation de la prolifération cellulaire et la réparation de l’ADN. Le régulateur transcriptionnel BAP1, une déubiquitinase nucléaire, a été initialement identifié pour sa capacité à promouvoir la fonction suppressive de tumeurs de BRCA1. BAP1 forme un complexe multi-protéique avec plusieurs facteurs transcriptionnels et sa fonction principale est la déubiquitination de H2Aub. Plusieurs études ont démontré que BAP1 est un gène suppresseur de tumeurs majeur et qu’il est largement muté et inactivé dans une multitude de cancers. En effet, BAP1 émerge comme étant la DUB la plus mutée au niveau des cancers. Cependant, le ou les mécanismes d’action et de régulation du complexe BAP1 restent très peu connus. Dans cette étude nous nous sommes intéressés à la caractérisation moléculaire et fonctionnelle des partenaires protéiques de BAP1. De manière significative nous avons caractérisé un mécanisme unique de régulation entre deux composants majeurs du complexe BAP1 à savoir, HCF-1 et OGT. En effet, nous avons démontré que HCF-1 est requis pour maintenir le niveau protéique de OGT et que cette dernière est indispensable pour la maturation protéolytique de HCF-1 en promouvant son clivage par O-GlcNAcylation, une signalisation cellulaire nécessaire au bon fonctionnement de HCF-1. Également, nous avons découvert un nouveau mécanisme de régulation de BAP1 par l’ubiquitine ligase atypique UBE2O. En effet, UBE2O agit comme un régulateur négatif de BAP1 puisque l’ubiquitination de ce dernier induit sa séquestration dans le cytoplasme et l’inhibition de sa fonction suppressive de tumeurs. D’autre part nous nous sommes penchés sur la caractérisation de l’association de BAP1 avec deux facteurs de la famille des protéines Polycombes nommés ASXL1 et ASXL2 (ASXL1/2). Nous avons investigué le rôle de BAP1/ASXL1/2, particulièrement dans les mécanismes de déubiquitination et suppression de tumeurs. Nous avons démontré que BAP1 interagit directement iii via son domaine C-terminale avec le même domaine ASXM de ASXL1/2 formant ainsi deux complexes mutuellement exclusifs indispensables pour induire l’activité déubiquitinase de BAP1. De manière significative, ASXM s’associe avec BAP1 pour créer un nouveau domaine composite de liaison à l’ubiquitine. Ces interactions BAP1/ASXL1/2 régulent la progression harmonieuse du cycle cellulaire. De plus, la surexpression de BAP1 et de ASXL2 au niveau des fibroblastes induit la sénescence de manière dépendante de leurs interactions. D’autre part, nous avons identifié des mutations de cancers au niveau de BAP1 le rendant incapable de lier ASXL1/2, d’exercer sa fonction d’autodéubiquitination et de ce fait d’agir comme suppresseur de tumeurs. Ainsi nous avons révélé un lien étroit entre le gène suppresseur de tumeurs BAP1, son activité déubiquitinase et le contrôle de la prolifération cellulaire.

Flower abscission and fruit set on table grapes (Vitis vinifera L.): unraveling physiological and molecular mechanisms

Doutoramento em Engenharia Agronómica - Instituto Superior de Agronomia - UL

ANALYSIS OF POPULUS TREMULOIDES CLONAL VARIATION AND DELINEATION IN THE OTTAWA NATIONAL FOREST

The technique of delineating Populus tremuloides (Michx.) clonal colonies based on morphology and phenology has been utilized in many studies and forestry applications since the 1950s. Recently, the availability and robustness of molecular markers has challenged the validity of such approaches for accurate clonal identification. However, genetically sampling an entire stand is largely impractical or impossible. For that reason, it is often necessary to delineate putative genet boundaries for a more selective approach when genetically analyzing a clonal population. Here I re-evaluated the usefulness of phenotypic delineation by: (1) genetically identifying clonal colonies using nuclear microsatellite markers, (2) assessing phenotypic inter- and intraclonal agreement, and (3) determining the accuracy of visible characters to correctly assign ramets to their respective genets. The long-term soil productivity study plot 28 was chosen for analysis and is located in the Ottawa National Forest, MI (46° 37'60.0" N, 89° 12'42.7" W). In total, 32 genets were identified from 181 stems using seven microsatellite markers. The average genet size was 5.5 ramets and six of the largest were selected for phenotypic analyses. Phenotypic analyses included budbreak timing, DBH, bark thickness, bark color or brightness, leaf senescence, leaf serrations, and leaf length ratio. All phenotypic characters, except for DBH, were useful for the analysis of inter- and intraclonal variation and phenotypic delineation. Generally, phenotypic expression was related to genotype with multiple response permutation procedure (MRPP) intraclonal distance values ranging from 0.148 and 0.427 and an observed MRPP delta value=0.221 when the expected delta=0.5. The phenotypic traits, though, overlapped significantly among some clones. When stems were assigned into phenotypic groups, six phenotypic groups were identified with each group containing a dominant genotype or clonal colony. All phenotypic groups contained stems from at least two clonal colonies and no clonal colony was entirely contained within one phenotypic group. These results demonstrate that phenotype varies with genotype and stand clonality can be determined using phenotypic characters, but phenotypic delineation is less precise. I therefore recommend that some genetic identification follow any phenotypic delineation. The amount of genetic identification required for clonal confirmation is likely to vary based on stand and environmental conditions. Further analysis, however, is needed to test these findings in other forest stands and populations.

Culturas celulares

As culturas celulares apareceram no século XX e têm vindo a evoluir como método de estudo e análise de patologias e de novos medicamentos para as combater. Com o passar dos anos surgiram diferentes métodos de cultivo de células diversificando assim as culturas celulares em mono-camada, suspensas e imobilizadas. Um dos propósitos das culturas celulares é a manipulação das células de modo a permitir a visualização das alterações celulares como o envelhecimento da célula (senescência), a apoptose, a necrose e a autofagia da célula. Este método tem provado ser útil tanto no diagnostico e tratamento de várias patologias como em doenças oncológicas, neuro-degenerativas ou do sistema imunitário.