Sea bream (Sparus aurata) intensive larval rearing : effects of microalgae and phages addition on the performance of the larvae

Máster Oficial en Cultivos Marinos. Trabajo presentado como requisito parcial para la obtención del Título de Máster Oficial en Cultivos Marinos, otorgado por la Universidad de Las Palmas de Gran Canaria (ULPGC), el Instituto Canario de Ciencias Marinas (ICCM), y el Centro Internacional de Altos Estudios Agronómicos Mediterráneos de Zaragoza (CIHEAM)

Design, realization and characterization of automated millifluidic bioreactors for investigating the molecular evolution of lytic or lysogenic vector phages infecting engineered host e. Coli

In questa tesi viene presentato un bioreattore in grado di mantenere nel tempo condizioni biologiche tali che consentano di massimizzare i cicli di evoluzione molecolare di vettori di clonazione fagici: litico (T7) o lisogeno (M13). Verranno quindi introdtti concetti legati alla Teoria della Quasispecie e alla relazione tra errori di autoreplicazione e pressioni selettive naturali o artificiali su popolazioni di virus: il modello naturale del sistema evolutivo. Tuttavia, mantenere delle popolazioni di virus significa formire loro un substrato dove replicare. Per fare ciò, altri gruppi di ricerca hanno giá sviluppato complessi e costosi prototipi di macchinari per la crescita continua di popolazioni batteriche: i compartimenti dei sistemi evolutivi. Il bioreattore, oggetto di questo lavoro, fa parte del progetto europeo Evoprog: general purpose programmable machine evolution on a chip (Jaramillo’s Lab, University of Warwick) che, utilizzando tecnologie fagiche e regolazioni sintetiche esistenti, sará in grado di produrre funzionalità biocomputazionali di due ordini di grandezza più veloci rispetto alle tecniche convenzionali, riducendo allo stesso tempo i costi complessivi. Il primo prototipo consiste in uno o piú fermentatori, dove viene fatta crescere la cultura batterica in condizioni ottimizzate di coltivazione continua, e in un cellstat, un volume separato, dove avviene solo la replicazione dei virus. Entrambi i volumi sono di pochi millilitri e appropriatamente interconnessi per consentire una sorta di screening continuo delle biomolecole prodotte all’uscita. Nella parte finale verranno presentati i risultati degli esperimenti preliminari, a dimostrazione dell’affidabilità del prototipo costruito e dei protocolli seguiti per la sterilizzazione e l’assemblaggio del bioreattore. Gli esperimenti effettuati dimostrano il successo di due coltivazioni virali continue e una ricombinazione in vivo di batteriofagi litici o lisogeni ingegnerizzati. La tesi si conclude valutando i futuri sviluppi e i limiti del sistema, tenendo in considerazione, in particolare, alcune applicazioni rivolte agli studi di una terapia batteriofagica.

The basel cocktail for simultaneous phenotyping of human cytochrome P450 isoforms in plasma, saliva and dried blood spots.

BACKGROUND AND OBJECTIVE Phenotyping cocktails use a combination of cytochrome P450 (CYP)-specific probe drugs to simultaneously assess the activity of different CYP isoforms. To improve the clinical applicability of CYP phenotyping, the main objectives of this study were to develop a new cocktail based on probe drugs that are widely used in clinical practice and to test whether alternative sampling methods such as collection of dried blood spots (DBS) or saliva could be used to simplify the sampling process. METHODS In a randomized crossover study, a new combination of commercially available probe drugs (the Basel cocktail) was tested for simultaneous phenotyping of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4. Sixteen subjects received low doses of caffeine, efavirenz, losartan, omeprazole, metoprolol and midazolam in different combinations. All subjects were genotyped, and full pharmacokinetic profiles of the probe drugs and their main metabolites were determined in plasma, dried blood spots and saliva samples. RESULTS The Basel cocktail was well tolerated, and bioequivalence tests showed no evidence of mutual interactions between the probe drugs. In plasma, single timepoint metabolic ratios at 2 h (for CYP2C19 and CYP3A4) or at 8 h (for the other isoforms) after dosing showed high correlations with corresponding area under the concentration-time curve (AUC) ratios (AUC0-24h parent/AUC0-24h metabolite) and are proposed as simple phenotyping metrics. Metabolic ratios in dried blood spots (for CYP1A2 and CYP2C19) or in saliva samples (for CYP1A2) were comparable to plasma ratios and offer the option of minimally invasive or non-invasive phenotyping of these isoforms. CONCLUSIONS This new combination of phenotyping probe drugs can be used without mutual interactions. The proposed sampling timepoints have the potential to facilitate clinical application of phenotyping but require further validation in conditions of altered CYP activity. The use of DBS or saliva samples seems feasible for phenotyping of the selected CYP isoforms.

An extensive repertoire of type III secretion effectors in Escherichia coli O157 and the role of lambdoid phages in their dissemination

Several pathogenic strains of Escherichia coli exploit type III secretion to inject effector proteins into human cells, which then subvert eukaryotic cell biology to the bacterium's advantage. We have exploited bioinformatics and experimental approaches to establish that the effector repertoire in the Sakai strain of enterohemorrhagic E. coli (EHEC) O157:H7 is much larger than previously thought. Homology searches led to the identification of > 60 putative effector genes. Thirteen of these were judged to be likely pseudogenes, whereas 49 were judged to be potentially functional. In total, 39 proteins were confirmed experimentally as effectors: 31 through proteomics and 28 through translocation assays. At the protein level, the EHEC effector sequences fall into > 20 families. The largest family, the NleG family, contains 14 members in the Sakai strain alone. EHEC also harbors functional homologs of effectors from plant pathogens (HopPtoH, HopW, AvrA) and from Shigella (OspD, OspE, OspG), and two additional members of the Map/IpgB family. Genes encoding proven or predicted effectors occur in > 20 exchangeable effector loci scattered throughout the chromosome. Crucially, the majority of functional effector genes are encoded by nine exchangeable effector loci that lie within lambdoid prophages. Thus, type III secretion in E. coli is linked to a vast phage metagenome, acting as a crucible for the evolution of pathogenicity.

Mixing the grant cocktail:towards an understanding of the outcomes of financial support to small firms

One of the key policy objectives of government at national and regional level, is to overcome the constraints preventing local industry achieving greater competitiveness in the international market-place. This paper examines the impact of grant assistance to Northern Ireland small firms delivered over the period 1994 ^ 97 by the former Local Enterprise Development Unit through its Growth Business Support Programme (GBSP). Previous work by the authors showed that there was some tentative evidence to suggest a link between employment growth and grant aid provided to very small firms (fewer than 10 employees) assisted under the GBSP. The central objective of the empirical work reported in this paper is to extend the previous analysis by understanding the extent to which the value of financial assistance influences growth (employment, turnover, and productivity measures) and if differential impacts arise depending on the nature and timing (lag structures) of the grant assistance.

Fate of Surface-Inoculated Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium on Kippered Beef during Extended Storage at Refrigeration and Abusive Temperatures

The behavior of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium on kippered beef was evaluated. Individual pieces of the product were separately inoculated on the top and bottom surfaces with each three- to six-strain pathogen cocktail at ca. 6.0 log CFU per piece and stored at 4, 10, 21, or 30 degrees C for up to 28 days in each of two trials. When kippered beef was inoculated with E coli O157:H7, Salmonella Typhimurium, or L. monocytogenes and stored at 4, 10, 2 1, or 30 degrees C for up to 28 days, pathogen numbers decreased ca. 0.4 to 0.9, 1.0 to 1.8, 3.0 to >= 5.25, and >= 5.0 to 5.25 log CFU per piece, respectively. Average D-values for E. coli O157:H7, Salmonella Typhimurium, and L. monocytogenes stored at 4 to 30 degrees C for 28 days were ca. 41 to 4.6, 40.8 to 5.3, and 29.5 to 4.3 days, respectively. As expected, the higher the storage temperature, the greater the level and rate of inactivation for all three pathogens. These data establish that kippered beef does not provide an environment conducive to proliferation of these pathogens.

Effect of inulin as prebiotic and synbiotic interactions between probiotics to improve fermented milk firmness

Inulin behaved as a prebiotic to improve firmness of skim milk fermented by (a) pure cultures of Lactobacillus acidophilus (La), Lactobacillus rhamnosus (Lr), Lactobacillus bulgaricus (Lb) and Bifidobacterium lactis (Bl), (b) binary co-cultures of them with Streptococcus thermophilus (St), or (c) a cocktail containing all them. Inulin addition to co-cultures and cocktail enhanced products firmness, either after 1 day (D1) or 7 days (D7) of cold storage, likely due to the increase in microbial growth induced by metabolic interactions among lactic acid bacteria and partial inulin metabolization. Co-culture firmness did in fact range from 0.33 N without inulin (St-Lb) after D1 and 0.55 N with inulin (St-Lr) after D7. Also cocktail cultures exhibited high values of firmness, ranging, as an average, from 0.43 N (D1) to 0.46 N (D7), which suggests that they could have been potentiated by the reciprocal synergistic effects of microorganisms in complex mixture. (C) 2011 Elsevier Ltd. All rights reserved.

Effect of inulin on growth and acidification performance of different probiotic bacteria in co-cultures and mixed culture with Streptococcus thermophilus

Inulin was used as a prebiotic to improve the quality and consistency of skim milk fermented by Lactobacillus acidophilus (La), Lactobacillus rhamnosus (Lr), Lactobacillus bulgaricus (Lb) and Bifidobacterium lactis (BI) with Streptococcus thermophilus (St), either in binary co-cultures or in cocktail containing all microorganisms. We compared, either in the presence of 40 mg inulin g(-1) or not, the results of the maximum acidification rate (V(max)) and the times to reach it (t(max)), to reach pH 5.0 (t(PH5.0)) and to complete the fermentation (t(f)). Post-acidification, lactic acid formation and cell counts were also compared after either 1 day (D1) or 7 days of storage at 4 degrees C (N). In co-culture, inulin addition to the milk increased V(max), decreased t(max) and t(f), favored post-acidification and exerted a bifidogenic effect. S. thermophilus proved to stimulate the metabolism of the other lactic bacteria and enhanced the product features. After D7, a significant prebiotic effect of inulin was observed in all co-cultures. Either after D1 or D7, the enumerations of Lr and BI in mixed culture markedly decreased compared to their respective co-cultures because of greater competition for the same substrates. (C) 2008 Elsevier Ltd. All rights reserved.

Soybean growth and yield after single tillage and species mixture of cover plants

The use of cover crops is important for the agricultural crop and soil management in order to improve the system and, consequently, to increase yield. Therefore, the present study analyzed the effect of crop residues of black oat (Avena strigosa Schreb.) (BO) and a cocktail (CO) of BO, forage turnip (Raphanus sativus L.) (FT) and common vetch (Vicia sativa L.) (V) on the emergence speed index (ESI), seedling emergence speed (SES) plant height and soybean yield in different intervals between cover crop desiccation with glyphosate 480 (3 L ha-1) and BRS 232 cultivar sowing. Plots of 5 x 2.5 m with 1 m of border received four treatments with BO cover crops and four with CO as well as a control for each cover crop, at random, with five replications. The plots were desiccated in intervals of 1, 10, 20 and 30 days before soybean seeding. The harvest was manual while yield was adjusted to 13% of moisture content. The experimental design was completely randomized with splitplots and means compared by the Scott and Knott test at 5% of significance. The results showed that CO of cover crops can be recommended for soybean to obtain a more vigorous seedling emergence, from 10 days after cover crop desiccation.

Optimizing potentiometric ionophore and electrode design for environmental on-site control of antibiotic drugs: Application to sulfamethoxazole

Potentiometric sensors are typically unable to carry out on-site monitoring of environmental drug contaminants because of their high limits of detection (LODs). Designing a novel ligand material for the target analyte and managing the composition of the internal reference solution have been the strategies employed here to produce for the first time a potentiometric-based direct reading method for an environmental drug contaminant. This concept has been applied to sulfamethoxazole (SMX), one of the many antibiotics used in aquaculture practices that may occur in environmental waters. The novel ligand has been produced by imprinting SMX on the surface of graphitic carbon nanostructures (CN) < 500 nm. The imprinted carbon nanostructures (ICN) were dispersed in plasticizer and entrapped in a PVC matrix that included (or not) a small amount of a lipophilic additive. The membrane composition was optimized on solid-contact electrodes, allowing near-Nernstian responses down to 5.2 μg/mL and detecting 1.6 μg/mL. The membranes offered good selectivity against most of the ionic compounds in environmental water. The best membrane cocktail was applied on the smaller end of a 1000 μL micropipette tip made of polypropylene. The tip was then filled with inner reference solution containing SMX and chlorate (as interfering compound). The corresponding concentrations were studied for 1 × 10−5 to 1 × 10−10 and 1 × 10−3 to 1 × 10−8 mol/L. The best condition allowed the detection of 5.92 ng/L (or 2.3 × 10−8 mol/L) SMX for a sub-Nernstian slope of −40.3 mV/decade from 5.0 × 10−8 to 2.4 × 10−5 mol/L.

Strategies to combat infections of Acinetobacter baumannii biofilms

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)

Selection of information in auditory virtual reality

ERP, auditory virtual reality, dichotic listening, selective auditory attention, cocktail-party phenomenon, HRTF

Predator-prey chemical warfare determines the expression of biocontrol genes by rhizosphere-associated Pseudomonas fluorescens.

Soil bacteria are heavily consumed by protozoan predators, and many bacteria have evolved defense strategies such as the production of toxic exometabolites. However, the production of toxins is energetically costly and therefore is likely to be adjusted according to the predation risk to balance the costs and benefits of predator defense. We investigated the response of the biocontrol bacterium Pseudomonas fluorescens CHA0 to a common predator, the free-living amoeba Acanthamoeba castellanii. We monitored the effect of the exposure to predator cues or direct contact with the predators on the expression of the phlA, prnA, hcnA, and pltA genes, which are involved in the synthesis of the toxins, 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin, hydrogen cyanide, and pyoluteorin, respectively. Predator chemical cues led to 2.2-, 2.0-, and 1.2-fold increases in prnA, phlA, and hcnA expression, respectively, and to a 25% increase in bacterial toxicity. The upregulation of the tested genes was related to the antiprotozoan toxicity of the corresponding toxins. Pyrrolnitrin and DAPG had the highest toxicity, suggesting that bacteria secrete a predator-specific toxin cocktail. The response of the bacteria was elicited by supernatants of amoeba cultures, indicating that water-soluble chemical compounds were responsible for induction of the bacterial defense response. In contrast, direct contact of bacteria with living amoebae reduced the expression of the four bacterial toxin genes by up to 50%, suggesting that protozoa can repress bacterial toxicity. The results indicate that predator-prey interactions are a determinant of toxin production by rhizosphere P. fluorescens and may have an impact on its biocontrol potential.