Tnf-a production and apoptosis in hepatocytes after listeria monocytogenes and salmonella typhimurium invasion

Invasion of hepatocytes by Listeria monocytogenes (LM) and Salmonella Typhimurium (ST) can stimulate tumor necrosis factor alpha (TNF-α) release and induce apoptosis. In this study, we compared the behavior of hepatocytes invaded by three L. monocytogenes serotypes (LM-4a, LM-4b and LM-1/2a) and by ST to understand which bacterium is more effective in the infectious process. We quantified TNF-α release by ELISA, apoptosis rates by annexin V (early apoptosis) and TUNEL (late apoptosis) techniques. The cell morphology was studied too. TNF-α release rate was highest in ST-invaded hepatocytes. ST and LM-1/2a induced the highest apoptosis production rates evaluated by TUNEL. LM-4b produced the highest apoptosis rate measured by annexin. Invaded hepatocytes presented various morphological alterations. Overall, LM-4b and LM-1/2a proved to be the most efficient at cell invasion, although ST adapted faster to the environment and induced earlier hepatocyte TNF-α release.

Production and Characterization of the biological activity of peptides obtained via hydrolysis from whey proteins by cardosins

Dissertation presented to obtain the Ph.D degree in Biochemistry, Engineering and Technological Sciences

Production and characterization of monoclonal antibodies to the edta extract of Leptospira interrogans, serovar icterohaemorrhagiae

Monoclonal antibodies (MABs) ivere produced against an etbylenediaminetetraacetate (EDTA) extract of Leptospira interrogans serovar icterohaemorrhagiae being characterized by gel precipitation as IgM and IgG (IgGl and IgG2b). The EDTA extract was detected as several bands by silver staining in SDS-PAGE. In the Western blot the bands around 20 KDa reacted with a monoclonal antibody, 47B4D6, and was oxidized by periodate and was not digested by pronase, suggesting that the determinant is of carbohydrate nature, lmmunocytochemistry, using colloidal gold labeling, showed that an EDTA extract determinant recognized by monoclonal antibody 47B4D6, is localized under the outer envelope of serovar icterohaemorrhagiae. Hoe AIAB raised against the EDTA extract was not able to protect hamsters from lethal challenge with virulent homologous leptospires.

Studies on the wine spoilage yeast Brettanomyces bruxellensis: 4-ethylphenol production and improved detection

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Production and diagnostic application of recombinant domain III of West Nile envelope protein in Brazil

INTRODUCTION: West Nile virus (WNV) is a flavivirus with a natural cycle involving mosquitoes and birds. Over the last 11 years, WNV has spread throughout the Americas with the imminent risk of its introduction in Brazil. METHODS: Envelope protein domain III of WNV (rDIII) was bacterially expressed and purified. An enzyme-linked immunosorbent assay with WNV rDIII antigen was standardized against mouse immune fluids (MIAFs) of different flavivirus. RESULTS: WNV rDIII reacted strongly with St. Louis encephalitis virus (SLEV) MIAF but not with other flaviviruses. CONCLUSIONS: This antigen may be a potentially useful tool for serologic diagnosis and may contribute in future epidemiological surveillance of WNV infections in Brazil.

Antifungal susceptibility and distribution of Candida spp. isolates from the University Hospital in the municipality of Dourados, State of Mato Grosso do Sul, Brazil

Introduction Hospital infections caused by Candida spp. are a leading cause of morbidity and mortality in hospitalized patients, particularly those that are critically ill or immunocompromised. In this study, the distribution of Candida species in isolates from the University Hospital of the Federal University at Grande Dourados and their in vitro susceptibility to antifungal drugs were analyzed. Methods Yeasts were phenotypically identified using classical methodologies. Antifungal susceptibility tests to amphotericin B and fluconazole were performed using the broth microdilution technique. Results A total of 50 Candida isolates were obtained from hospitalized patients during the study period. We analyzed yeast isolates from urine (n=31; 62%), blood (n=12; 24%), and tracheal secretions (n=7; 14%). The following Candida species were identified: C. tropicalis (n=21; 42%), C. albicans (n=18; 36%), C. glabrata (n=10; 20%), and C. krusei (n=1; 2%). Antifungal susceptibility tests demonstrated that C. albicans was susceptible to both antifungal agents. However, 31.2% of the non-C. albicans Candida isolates displayed dose-dependent susceptibility to fluconazole, and 3.1% were resistant to amphotericin B. Conclusions In contrast to previous reports, our results indicated that C. tropicalis was the most commonly isolated yeast species among the hospital patients. The predominance of non-C. albicans Candida infections confirms the importance of species-level identification for implementing appropriate antifungal therapies.

Production and characterization of Chitin-Glucan Complex by Komagataella pastoris

This thesis was focused on the production, extraction and characterization of chitin:β-glucan complex (CGC). In this process, glycerol byproduct from the biodiesel industry was used as carbon source. The selected CGC producing yeast was Komagataella pastoris (formerly known as Pichia pastoris), due the fact that to achieved high cell densities using as carbon source glycerol from the biodiesel industry. Firstly, a screening of K. pastoris strains was performed in shake flask assays, in order to select the strain of K. pastoris with better performance, in terms of growth, using glycerol as a carbon source. K. pastoris strain DSM 70877 achieved higher final cell densities (92-97 g/l), using pure glycerol (99%, w/v) and in glycerol from the biodiesel industry (86%, w/v), respectively, compared to DSM 70382 strain (74-82 g/l). Based on these shake flask assays results, the wild type DSM 70877 strain was selected to proceed for cultivation in a 2 l bioreactor, using glycerol byproduct (40 g/l), as sole carbon source. Biomass production by K. pastoris was performed under controlled temperature and pH (30.0 ºC and 5.0, respectively). More than 100 g/l biomass was obtained in less than 48 h. The yield of biomass on a glycerol basis was 0.55 g/g during the batch phase and 0.63 g/g during the fed-batch phase. In order to optimize the downstream process, by increasing extraction and purification efficiency of CGC from K. pastoris biomass, several assays were performed. It was found that extraction with 5 M NaOH at 65 ºC, during 2 hours, associated to neutralization with HCl, followed by successive washing steps with deionised water until conductivity of ≤20μS/cm, increased CGC purity. The obtained copolymer, CGCpure, had a chitin:glucan molar ratio of 25:75 mol% close to commercial CGC samples extracted from A. niger mycelium, kiOsmetine from Kitozyme (30:70 mol%). CGCpure was characterized by solid-state Nuclear Magnetic Resonance (NMR) spectroscopy and Differential Scanning Calorimetry (DCS), revealing a CGC with higher purity than a CGC commercial (kiOsmetine). In order to optimize CGC production, a set of batch cultivation experiments was performed to evaluate the effect of pH (3.5–6.5) and temperature (20–40 ºC) on the specific cell growth rate, CGC production and polymer composition. Statistical tools (response surface methodology and central composite design) were used. The CGC content in the biomass and the volumetric productivity (rp) were not significantly affected within the tested pH and temperature ranges. In contrast, the effect of pH and temperature on the CGC molar ratio was more pronounced. The highest chitin: β-glucan molar ratio (> 14:86) was obtained for the mid-range pH (4.5-5.8) and temperatures (26–33 ºC). The ability of K. pastoris to synthesize CGC with different molar ratios as a function of pH and temperature is a feature that can be exploited to obtain tailored polymer compositions.(...)

Production and characterization of electrospun composite fibers: confinement of thermosensitive microgels

Materials engineering focuses on the assembly of materials´ properties to design new products with the best performance. By using sub-micrometer size materials in the production of composites, it is possible to obtain objects with properties that none of their compounds show individually. Once three-dimensional materials can be easily customized to obtain desired properties, much interest has been paid to nanostructured poly-mers in order to build biocompatible devices. Over the past years, the thermosensitive microgels have become more common in the framework of bio-materials with potential applicability in therapy and/or diagnostics. In addition, high aspect ratio biopolymers fibers have been produced using the cost-effective method called electrospinning. Taking advantage of both microgels and electrospun fibers, surfaces with enhanced functionalities can be obtained and, therefore employed in a wide range of applications. This dissertation reports on the confinement of stimuli-responsive microgels through the colloidal electro-spinning process. The process mainly depends on the composition, properties and patterning of the precur-sor materials within the polymer jet. Microgels as well as the electrospun non-woven mats were investigated to correlate the starting materials with the final morphology of the composite fibers. PNIPAAm and PNIPAAm/Chitosan thermosensitive microgels with different compositions were obtained via surfactant free emulsion polymerization (SFEP) and characterized in terms of chemical structure, morphology, thermal sta-bility, swelling properties and thermosensitivity. Finally, the colloidal electrospinning method was carried out from spinning solutions composed of the stable microgel dispersions (up to a concentration of about 35 wt. % microgels) and a polymer solution of PEO/water/ethanol mixture acting as fiber template solution. The confinement of microgels was confirmed by Scanning Electron Microscopy (SEM). The electrospinning process was statistically analysed providing the optimum set of parameters aimed to minimize the fiber diameter, which give rise to electrospun nanofibers of PNIPAAm microgels/PEO with a mean fiber diameter of 63 ± 25 nm.

Jeronimo Martins in 2030 succession and strategy planning

The case outlines the history of Jerónimo Martins and the Dos Santos family in the context of a dialogue between the pater familias Alexandre and his successor Pedro. The case idea analyses the problems associated with next generation entrance in the family business and the outcomes on the different stakeholders and environment, with a particular focus on the evolution of the relationship between family, family holding and the JM group. The case is designed to assess the sustainability of a traditional succession strategy in the context of the challenges of globalization and growth of the company and family, analysing the reciprocal influence in a long-term strategy.

Engineering of fatty acid production and secretion in Saccharomyces cerevisiae

Tese de Doutoramento em Ciências - Especialidade em Biologia

Measurements of Higgs boson production and couplings in the four-lepton channel in pp collisions at center-of-mass energies of 7 and 8 TeV with the ATLAS detector

The final ATLAS Run 1 measurements of Higgs boson production and couplings in the decay channel H→ZZ∗→ℓ+ℓ−ℓ′+ℓ′−, where ℓ,ℓ′=e or μ, are presented. These measurements were performed using pp collision data corresponding to integrated luminosities of 4.5 fb−1 and 20.3 fb−1 at center-of-mass energies of 7 TeV and 8 TeV, respectively, recorded with the ATLAS detector at the LHC. The H→ZZ∗→4ℓ signal is observed with a significance of 8.1 standard deviations at 125.36 GeV, the combined ATLAS measurement of the Higgs boson mass from the H→γγ and H→ZZ∗→4ℓ channels. The production rate relative to the Standard Model expectation, the signal strength, is measured in four different production categories in the H→ZZ∗→4ℓ channel. The measured signal strength, at this mass, and with all categories combined, is 1.44 +0.40−0.33. The signal strength for Higgs boson production in gluon fusion or in association with tt¯ or bb¯ pairs is found to be 1.7 +0.5−0.4, while the signal strength for vector-boson fusion combined with WH/ZH associated production is found to be 0.3 +1.6−0.9.

Systematics, functional morphology and distribution of a bivalve (Apachecorbula muriatica gen. et sp. nov.) from the rim of the ‘Valdivia Deep’ brine pool in the Red Sea

The deep brine pools of the Red Sea comprise extreme, inhospitable habitats yet house microbial communities that potentially may fuel adjacent fauna. We here describe a novel bivalve from a deep-sea (1525 m) brine pool in the Red Sea, where conditions of high salinity, lowered pH, partial anoxia and high temperatures are prevalent. Remotely operated vehicle (ROV) footage showed that the bivalves were present in a narrow (20 cm) band along the rim of the brine pool, suggesting that it is not only tolerant of such extreme conditions but is also limited to them. The bivalve is a member of the Corbulidae and named Apachecorbula muriatica gen. et sp. nov. The shell is atypical of the family in being modioliform and thin. The semi-infaunal habit is seen in ROV images and reflected in the anatomy by the lack of siphons. The ctenidia are large and typical of a suspension feeding bivalve, but the absence of guard cilia and the greatly reduced labial palps suggest that it is non-selective as a response to low food availability. It is proposed that the low body mass observed is a consequence of the extreme habitat and low food availability. It is postulated that the observed morphology of Apachecorbula is a result of paedomorphosis driven by the effects of the extreme environment on growth but is in part mitigated by the absence of high predation pressures.

Density, size and distribution of stomata in 35 rainforest tree species in Central Amazonia

Stomata are turgor-operated valves that control water loss and CO2 uptake during photosynthesis, and thereby water relation and plant biomass accumulation is closely related to stomatal functioning. The aims of this work were to document how stomata are distributed on the leaf surface and to determine if there is any significant variation in stomatal characteristics among Amazonian tree species, and finally to study the relationship between stomatal density (S D) and tree height. Thirty five trees (>17 m tall) of different species were selected. Stomatal type, density (S D), size (S S) and stomatal distribution on the leaf surface were determined using nail polish imprints taken from both leaf surfaces. Irrespective of tree species, stomata were located only on the abaxial surface (hypostomaty), with large variation in both S D and S S among species. S D ranged from 110 mm-2 in Neea altissima to 846 mm-2 in Qualea acuminata. However, in most species S D ranges between 271 and 543 mm-2, with a negative relationship between S D and S S. We also found a positive relationship between S D and tree height (r² = 0.14, p < 0.01), but no correlation was found between S D and leaf thickness. The most common stomatal type was anomocytic (37%), followed by paracytic (26%) and anisocytic (11%). We conclude that in Amazonian tree species, stomatal distribution on the leaf surface is a response most likely dependent on the genetic background of every species, rather than a reaction to environmental changes, and that somehow S D is influenced by environmental factors dependent on tree height.

Biomass production and essential oil yield from leaves, fine stems and resprouts using pruning the crown of Aniba canelilla (H.B.K.) (Lauraceae) in the Central Amazon

Aniba canelilla (H.B.K.) Mez. is a tree species from Amazon that produces essential oil. The oil extraction from its leaves and stems can be an alternative way to avoid the tree cutting for production of essential oil. The aim of this study was to analyse factors that may influence the essential oil production and the biomass of resprouts after pruning the leaves and stems of A. canelilla trees. The tree crowns were pruned in the wet season and after nine months the leaves and stems of the remaining crown and the resprouts were collected, in the dry season. The results showed that the essential oil yield and chemical composition differed among the stems, leaves and resprouts. The stems' essential oil production differed between the seasons and had a higher production in the resprouting stems than the old stems of the remaining crown. The production of essential oil and leaf biomass of resprouts were differently related to the canopy openness, indicating that light increases the production of the essential oil and decreases the biomass of resprouting leaves. This study revealed that plant organs differ in their essential oil production and that the canopy openness must be taken into account when pruning the A. canelilla tree crown in order to achieve higher oil productivity.