Insights into the role of almond CBF transcription factors in the environmental control of cold acclimation and dormancy break

Dissertation presented to obtain the Ph.D degree in Biology

Size-Control Regulation during Regeneration

Functional regeneration of organs upon injury is a key process for animals survival. Contrary to humans, some vertebrates are remarkably competent in regenerating after acute organ or appendage lesions. This advantageous skill allows overcoming limitations in repair even in adult stages, when tissues are fully developed, via a process of epimorphic regeneration. One such organism is the zebrafish, which can regenerate several organs, namely its heart, retina, spinal cord and fins. (...)

The role of Grainy head in epithelial tissue growth

Part of the results of this thesis was presented in the following meetings: Susana Ponte, Lara Carvalho, Inês Cristo and António Jacinto. The role of Grainy head in epithelial tissue growth. Drostuga 2013. Faro, Portugal, January 3rd 2014 [poster] Susana Ponte, Lara Carvalho, Inês Cristo and António Jacinto. The role of Grainy head in epithelial tissue growth. Drostuga 2014. Tomar, Portugal, September 5th-6th 2014 [poster]

AGNI: an API for the control of automomous service robots

With the continuum growth of Internet connected devices, the scalability of the protocols used for communication between them is facing a new set of challenges. In robotics these communications protocols are an essential element, and must be able to accomplish with the desired communication. In a context of a multi-­‐‑agent platform, the main types of Internet communication protocols used in robotics, mission planning and task allocation problems will be revised. It will be defined how to represent a message and how to cope with their transport between devices in a distributed environment, reviewing all the layers of the messaging process. A review of the ROS platform is also presented with the intent of integrating the already existing communication protocols with the ServRobot, a mobile autonomous robot, and the DVA, a distributed autonomous surveillance system. This is done with the objective of assigning missions to ServRobot in a security context.

Development of process control strategies exploiting knowledge from systems biology: application to MDCK suspension cells

Madine Darby Canine Kidney (MDCK) cell lines have been extensively evaluated for their potential as host cells for influenza vaccine production. Recent studies allowed the cultivation of these cells in a fully defined medium and in suspension. However, reaching high cell densities in animal cell cultures still remains a challenge. To address this shortcoming, a combined methodology allied with knowledge from systems biology was reported to study the impact of the cell environment on the flux distribution. An optimization of the medium composition was proposed for both a batch and a continuous system in order to reach higher cell densities. To obtain insight into the metabolic activity of these cells, a detailed metabolic model previously developed by Wahl A. et. al was used. The experimental data of four cultivations of MDCK suspension cells, grown under different conditions and used in this work came from the Max Planck Institute, Magdeburg, Germany. Classical metabolic flux analysis (MFA) was used to estimate the intracellular flux distribution of each cultivation and then combined with partial least squares (PLS) method to establish a link between the estimated metabolic state and the cell environment. The validation of the MFA model was made and its consistency checked. The resulted PLS model explained almost 70% of the variance present in the flux distribution. The medium optimization for the continuous system and for the batch system resulted in higher biomass growth rates than the ones obtained experimentally, 0.034 h-1 and 0.030 h-1, respectively, thus reducing in almost 10 hours the duplication time. Additionally, the optimal medium obtained for the continuous system almost did not consider pyruvate. Overall the proposed methodology seems to be effective and both proposed medium optimizations seem to be promising to reach high cell densities.

Tambaqui growth and survival when exposed to different photoperiods

The use of different photoperiods (light) were investigated during tambaqui (Colossoma macropomum) juvenile growth under captivity. Light intensity tested was: continuous dark (24hrs without light), natural photoperiod simulation (10hrs of light and 14hrs without light) and continuous light (24 with light). No mortality was recorded among treatments. Significant differences was observed after 50 days of experiment among mean fish weight, fish kept under a continuous darkness showed a better specific growth rate (6.02%) when compared to control fish (natural photo period, 3.67%). Fish exposed to continuous light presented the lowest mean specific growth rate (2.04%). It is possible to improve tambaqui juvenile weight gain performance when kept under continuous darkness.

Myeloid Sirtuin 2 expression does not impact long-term Mycobacterium tuberculosis control

Sirtuins (Sirts) regulate several cellular mechanisms through deacetylation of several transcription factors and enzymes. Recently, Sirt2 was shown to prevent the development of inflammatory processes and its expression favors acute Listeria monocytogenes infection. The impact of this molecule in the context of chronic infections remains unknown. We found that specific Sirt2 deletion in the myeloid lineage transiently increased Mycobacterium tuberculosis load in the lungs and liver of conditional mice. Sirt2 did not affect long-term infection since no significant differences were observed in the bacterial burden at days 60 and 120 post-infection. The initial increase in M. tuberculosis growth was not due to differences in inflammatory cell infiltrates in the lung, myeloid or CD4+ T cells. The transcription levels of IFN-?, IL-17, TNF, IL-6 and NOS2 were also not affected in the lungs by Sirt2-myeloid specific deletion. Overall, our results demonstrate that Sirt2 expression has a transitory effect in M. tuberculosis infection. Thus, modulation of Sirt2 activity in vivo is not expected to affect chronic infection with M. tuberculosis.

IL-17A promotes intracellular growth of Mycobacterium by inhibiting apoptosis of infected macrophages

The fate of infected macrophages is a critical aspect of immunity to mycobacteria. By depriving the pathogen of its intracellular niche, apoptotic death of the infected macrophage has been shown to be an important mechanism to control bacterial growth. Here, we show that IL-17 inhibits apoptosis of Mycobacterium bovis BCG- or Mycobacterium tuberculosis-infected macrophages thus hampering their ability to control bacterial growth. Mechanistically, we show that IL-17 inhibits p53, and impacts on the intrinsic apoptotic pathway, by increasing the Bcl2 and decreasing Bax expression, decreasing cytochrome c release from the mitochondria, and inhibiting caspase-3 activation. The same effect of IL-17 was observed in infected macrophages upon blockade of p53 nuclear translocation. These results reveal a previously unappreciated role for the IL-17/p53 axis in the regulation of mycobacteria-induced apoptosis and can have important implications in a broad spectrum of diseases where apoptosis of the infected cell is an important host defense mechanism.

Zeolite-based nanomaterials for the control of opportunistic pathogenic microorganisms

Dissertação de mestrado em Applied Biochemistry (área de especialização em Biomedicine)

The production of vermicompost from dairy sludge and its value as a plant growth medium

Dairy sludge generated at Glanbia Ingredients Ltd., Kilkenny has up until now been landspread. This study investigated the feasibility of using earthworms to vermicompost the sludge as an alternative method of treatment. It was found that high levels of ammonia in the sludge led to earthworm fatality but that by manually aerating the sludge the ammonia could be volatilised or by adding zeolite the ammonia could be absorbed, thus solving the problem. In a medium scale trial, the earthworm species Dendrobaena veneta and Eisenia fetida dominated the polyculture. Earthworms grew and generated cocoons during vermicomposting. During vermicomposting no leachate was generated. Nutrient changes took place during vermicomposting. There were high levels of nitrate, increased calcium and sulphate in the vermicomposted dairy sludge. The amount of magnesium, potassium and chloride did not change, while phosphate was undetectable after vermicomposting. The levels of nitrate and phosphate were good indicators of the extent of vermicomposting. The vermicomposted dairy sludge provided improved growth and yields of radishes and barley compared to the dairy sludge and control. Compared to the vermicompost, the dairy sludge provided heavier ryegrass yields and more marigolds with larger flower diameters. Generally, it is the amount of phosphate in dairy sludge that dictates how much can be applied as a fertiliser on land. Vermicomposting reduced the amount of phosphate to an undetectable level but on the other hand created a problem of high nitrate levels. In a pot trial with grass grown in vermicompost the nitrate leached from the vermicompost. In field conditions the leaching of nitrate might occur and could cause an increased risk of contamination of groundwater and watercourses.

Effects of Growth Hormone on Cardiac Remodeling During Resistance Training in Rats

Abstract Background: Although the beneficial effects of resistance training (RT) on the cardiovascular system are well established, few studies have investigated the effects of the chronic growth hormone (GH) administration on cardiac remodeling during an RT program. Objective: To evaluate the effects of GH on the morphological features of cardiac remodeling and Ca2+ transport gene expression in rats submitted to RT. Methods: Male Wistar rats were divided into 4 groups (n = 7 per group): control (CT), GH, RT and RT with GH (RTGH). The dose of GH was 0.2 IU/kg every other day for 30 days. The RT model used was the vertical jump in water (4 sets of 10 jumps, 3 bouts/wk) for 30 consecutive days. After the experimental period, the following variables were analyzed: final body weight (FBW), left ventricular weight (LVW), LVW/FBW ratio, cardiomyocyte cross-sectional area (CSA), collagen fraction, creatine kinase muscle-brain fraction (CK-MB) and gene expressions of SERCA2a, phospholamban (PLB) and ryanodine (RyR). Results: There was no significant (p > 0.05) difference among groups for FBW, LVW, LVW/FBW ratio, cardiomyocyte CSA, and SERCA2a, PLB and RyR gene expressions. The RT group showed a significant (p < 0.05) increase in collagen fraction compared to the other groups. Additionally, the trained groups (RT and RTGH) had greater CK-MB levels compared to the untrained groups (CT and GH). Conclusion: GH may attenuate the negative effects of RT on cardiac remodeling by counteracting the increased collagen synthesis, without affecting the gene expression that regulates cardiac Ca2+ transport.

Attack of Potyphagotarsonemus tatus on cotton treated with growth retardants

The effects of growth retardants on infestation by Potyphagotarsonemus tatus (broad mite) on cotton (Gossypium hirsutum L. cv. IAC-17) plants was studied. Cotton plants were sprayed with (2-ch1oroethy1) trimethylammonium chloride (CCC) 250, 350 and 450 ppm, and with 1,1-dimethyl-piperidinium chloride (Pix) at concentrations of 84, 167 and 250 ppm. Growth retardants did not give effective control of Potyphagotarsone mus tatus but application of Pix 167 ppm showed a tendency to reduce mite attack.

Effects of nutrient foliar sprays containing growth regulators on productivity of peanut (Arachis hypogaea L. cv. Tatu-53)

The effects of the application of a macronutrient foliar spray combined with micronutrients and growth regulators (Unifol) on peanut grown in a soil with high fertility were investigated. A control without fertilizer and a soil fertilization (250 kg/ha) with NPK 9-30-16 were also established. Other treatments were as follows: Unifol fertilizer (18-12 16) applied 23 days after germination: Unifol (18-12-6) applied at the beginning of flowering; Unifol (18-12-6) applied during flowering, and Unifol (18-12-6) applied 23 days after germination plus Unifol (7-23-7) at the beginning of flowering. No significant differences were found amongst treatments, but certain treatments showed higher productivity e given Unifol fertilizer (18-12-6) applied 23 days after germination plus Unifol (7-23-7) at the flower anthesis. In this treatment, the number of pods, weight of seeds and production of seeds were higher. Best production of forage occurred in the treatment receiving soil fertilization.

Effects of both ecdysone and the acclimation to low temperature, on growth and metabolic rate of juvenile freshwater crayfish Cherax quadricarinatus (Decapoda, Parastacidae)

Growth, metabolic rate, and energy reserves of Cherax quadricarinatus (von Martens, 1868) juveniles were evaluated in crayfish acclimated for 16 weeks to either 25ºC (temperature near optimum) or 20ºC (marginal for the species). Additionally, the modulating effect of ecdsyone on acclimation was studied. After 12 weeks of exposure, weight gain of both experimental groups acclimated to 25ºC (control: C25, and ecdysone treated: E25) was significantly higher than that of those groups acclimated to 20ºC (C20 and E20). A total compensation in metabolic rate was seen after acclimation from 25ºC to 20ºC; for both the control group and the group treated with ecdysone. A Q10value significantly higher was only observed in the group acclimated to 20ºC and treated with ecdysone. A reduction of glycogen reserves in both hepatopancreas and muscle, as well as a lower protein content in muscle, was seen in both groups acclimated to 20ºC. Correspondingly, glycemia was always higher in these groups. Increased lipid levels were seen in the hepatopancreas of animals acclimated to 20ºC, while a higher lipid level was also observed in muscle at 20ºC, but only in ecdysone-treated crayfish.

Promise for plant pest control: root-associated pseudomonads with insecticidal activities.

Insects are an important and probably the most challenging pest to control in agriculture, in particular when they feed on belowground parts of plants. The application of synthetic pesticides is problematic owing to side effects on the environment, concerns for public health and the rapid development of resistance. Entomopathogenic bacteria, notably Bacillus thuringiensis and Photorhabdus/Xenorhabdus species, are promising alternatives to chemical insecticides, for they are able to efficiently kill insects and are considered to be environmentally sound and harmless to mammals. However, they have the handicap of showing limited environmental persistence or of depending on a nematode vector for insect infection. Intriguingly, certain strains of plant root-colonizing Pseudomonas bacteria display insect pathogenicity and thus could be formulated to extend the present range of bioinsecticides for protection of plants against root-feeding insects. These entomopathogenic pseudomonads belong to a group of plant-beneficial rhizobacteria that have the remarkable ability to suppress soil-borne plant pathogens, promote plant growth, and induce systemic plant defenses. Here we review for the first time the current knowledge about the occurrence and the molecular basis of insecticidal activity in pseudomonads with an emphasis on plant-beneficial and prominent pathogenic species. We discuss how this fascinating Pseudomonas trait may be exploited for novel root-based approaches to insect control in an integrated pest management framework.