NOR network transduction by generalized gate merging and substitution procedures : (principles of NOR network transduction programs NETTRA-G3 and NETTRA-G4) /

Includes bibliographical references.

Iron acquisition mechanisms of Flavobacterium psychrophilum

Forty strains of Flavobacterium psychrophilum were tested for the production of siderophores using the universal Chrome Azurol S (CAS) assay. The majority of the strains (85%) were CAS positive (CAS+) and some (15%) were CAS negative (CAS−). The cryptic plasmid pCP1 was carried by all positive strains and was lacking from negative strains. While a weak catechol reaction was detectable in CAS+ culture supernatants, the CAS reaction was, to some extent, heat sensitive, questioning whether the positive reaction was caused only by siderophores. The ability to grow in vitro under iron-restricted conditions did not correlate with the CAS reactivity, as growth of both CAS+ and CAS− strains was similarly impaired under iron restriction induced by 2,2 dipyridyl. Suppressed growth under these conditions was restored by addition of FeCl3, haemoglobin and transferrin for both CAS+ and CAS− strains.

Determinación de la contribución de los beneficios referidos al impuesto sobre la renta vigentes a 2014 en la incorporación de la responsabilidad social empresarial –RSE– a partir de la guía g4 de la iniciativa de reporte global –GRI– en las personas jurídicas de Colombia

La actividad empresarial en Colombia se desarrolla considerando factores que la condicionan siendo estos, sociales, políticos, económicos, ambientales, entre otros; motivo por el cual deben ser tenidos en cuenta en la gestión de las organizaciones. El modelo político colombiano garantiza la libertad de empresa, lo que se hace evidente en la jurisprudencia y doctrina, permitiéndoles desarrollar su actividad de manera regulada. Aunque-, la Constitución Nacional no señala de forma expresa la libertad de empresa, esta se puede colegir de lo contenido en el artículo 333 de dicho ordenamiento: “La actividad económica y la iniciativa privada son libres, dentro de los límites del bien común. Para su ejercicio, nadie podrá exigir permisos previos ni requisitos, sin autorización de la ley […]”.

Flavobacterium plurextorum sp. nov. Isolated from Farmed Rainbow Trout (Oncorhynchus mykiss)

Five strains (1126-1H-08(T), 51B-09, 986-08, 1084B-08 and 424-08) were isolated from diseased rainbow trout. Cells were Gram-negative rods, 0.7 µm wide and 3 µm long, non-endospore-forming, catalase and oxidase positive. Colonies were circular, yellow-pigmented, smooth and entire on TGE agar after 72 hours incubation at 25°C. They grew in a temperature range between 15°C to 30°C, but they did not grow at 37°Cor 42°C. Based on 16S rRNA gene sequence analysis, the isolates belonged to the genus Flavobacterium. Strain 1126-1H-08(T) exhibited the highest levels of similarity with Flavobacterium oncorhynchi CECT 7678(T) and Flavobacterium pectinovorum DSM 6368(T) (98.5% and 97.9% sequence similarity, respectively). DNA-DNA hybridization values were 87 to 99% among the five isolates and ranged from 21 to 48% between strain 1126-1H-08(T), selected as a representative isolate, and the type strains of Flavobacterium oncorhynchi CECT 7678(T) and other phylogenetic related Flavobacterium species. The DNA G+C content of strain 1126-1H-08(T) was 33.2 mol%. The predominant respiratory quinone was MK-6 and the major fatty acids were iso-C15∶0 and C15∶0. These data were similar to those reported for Flavobacterium species. Several physiological and biochemical tests differentiated the novel bacterial strains from related Flavobacterium species. Phylogenetic, genetic and phenotypic data indicate that these strains represent a new species of the genus Flavobacterium, for which the name Flavobacterium plurextorum sp. nov. was proposed. The type strain is 1126-1H-08(T) ( = CECT 7844(T) = CCUG 60112(T)).

Routines in a European aquaculture company and the development of a Flavobacterium psychrophilum dose response pre-model in adult Rainbow Trout

An internship in a European company dealing with aquaculture and biotechnology - AquaBioTech Group, Malta - was undertaken to complete the Master Degree of Science in Aquaculture of the School of Tourism and Maritime Technology of the Polytechnic Institute of Leiria. Biotechnology and aquaculture are two areas that have been synergistically used to contribute for the progress and improvement of fish production. The AquaBioTech Group is an example of a company able to integrate these areas to maximizing their services. Located in Mosta (Malta) the company operates in a sustainable way using Recirculation Aquaculture Systems (RAS) to maintain aquaculture species. In collaboration with several companies and institutions, the AquaBioTech Group is involved and supports the development of important international research projects. The present report focuses on two important parts of the internship performed during 6 months. Initially, it will cover the operation and constitution of the company, describing the routines and techniques acquired. Then, it will describe a pathology trial that forms the practical and scientific component of this report. Despite the limitation to describe some confidential assays, this trial consisted in the infection of Rainbow Trout (Oncorhynchus mykiss) with the bacterium Flavobacterium psychrophilum in order to evaluate the mortality rates over time. The internship served to solidify theoretical knowledge acquired during the academic training, develop professional skills and provide an understanding of jobs available on the market.

Pharmacokinetics and tumor disposition of PEGylated, methotrexate conjugated poly-l-lysine dendrimers

Dendrimers have potential for delivering chemotherapeutic drugs to solid tumours via the enhanced permeation and retention (EPR) effect. The impact of conjugation of hydrophobic anticancer drugs to hydrophilic PEGylated dendrimer surfaces, however, has not been fully investigated. The current study has therefore characterised the effect on dendrimer disposition of conjugating α-carboxyl protected methotrexate (MTX) to a series of PEGylated 3H-labelled poly-L-lysine dendrimers ranging in size from generation 3 (G3) to 5 (G5) in rats. Dendrimers contained 50% surface PEG and 50% surface MTX. Conjugation of MTX generally increased plasma clearance when compared to conjugation with PEG alone. Conversely, increasing generation reduced clearance, increased metabolic stability and reduced renal elimination of the administered radiolabel. For constructs with molecular weights >20 kDa increasing the molecular weight of conjugated PEG also reduced clearance and enhanced metabolic stability but had only a minimal effect on renal elimination. Tissue distribution studies revealed retention of MTX conjugated smaller (G3-G4) PEG570 dendrimers (or their metabolic products) in the kidneys. In contrast, the larger G5 dendrimer was concentrated more in the liver and spleen. The G5 PEG1100 dendrimer was also shown to accumulate in solid Walker 256 and HT1080 tumours and comparative disposition data in both rats (1 to 2% dose/g in tumour) and mice (11% dose/g in tumour) are presented. The results of this study further illustrate the potential utility of biodegradable PEGylated poly-L-lysine dendrimers as long circulating vectors for the delivery and tumour-targeting of hydrophobic drugs.

Interleukin 10 polymorphisms in ankylosing spondylitis

Genetic polymorphisms of the IL10 promoter region have been implicated in many autoimmune diseases, including seronegative spondyloarthropathies. We studied three SNPs (IL10-1087,-824, and -597) and two microsafellites(IL10R and IL10G) lying within the promoter region of IL10 for association with susceptibility to and clinical manifestations of ankylosing spondylitis (AS), a common form of spondyloarthritis. Four hundred and sixty-eight individuals from 182 Finnish families affected with AS were studied. No association between individual IL10 promoter region polymorphisms or marker haplotype was observed with susceptibility to AS, but weak association was noted between the IL10-597 and -824 SNPs and age of disease onset (P= 0.01 for each SNP). The IL10.G4 allele was associated with BASFI (corrected for disease duration) (P= 0.03). We conclude that IL10 promoter polymorphisms have no significant effect on susceptibility to AS, but may play a minor role in determining age of disease onset and disease severity. © 2003 Nature Publishing Group All rights reserved.

Preface

Abstract is not available.

Complexation of siRNA with Dendrimer: A Molecular Modeling Approach

This paper reports the structural behavior and thermodynamics of the complexation of siRNA with poly(amidoamine) (PAMAM) dendrimers of generation 3 (G3) and 4 (G4) through fully atomistic molecular dynamics (MD) simulations accompanied by free energy calculations and inherent structure determination. We have also done simulation with one siRNA and two dendrimers (2 x G3 or 2xG4) to get the microscopic picture of various binding modes. Our simulation results reveal the formation of stable siRNA-dendrimer complex over nanosecond time scale. With the increase in dendrimcr generation, the charge ratio increases and hence the binding energy between siRNA and dendrimer also increases in accordance with available experimental measurements. Calculated radial distribution functions of amines groups of various subgenerations in a given generation of dendrimer and phosphate in backbone of siRNA reveals that one dendrimer of generation 4 shows better binding with siRNA almost wrapping the dendrimer when compared to the binding with lower generation dendrimer like G3. In contrast, two dendrimers of generation 4 show binding without siRNA wrapping the den-rimer because of repulsion between two dendrimers. The counterion distribution around the complex and the water molecules in the hydration shell of siRNA give microscopic picture of the binding dynamics. We see a clear correlation between water. counterions motions and the complexation i.e. the water molecules and counterions which condensed around siRNA are moved away from the siRNA backbone when dendrimer start binding to the siRNA back hone. As siRNA wraps/bind to the dendrimer counterions originally condensed onto siRNA (Na-1) and dendrimer (Cl-) get released. We give a quantitative estimate of the entropy of counterions and show that there is gain in entropy due to counterions release during the complexation. Furthermore, the free energy of complexation of IG3 and IG4 at two different salt concentrations shows that increase in salt concentration leads to the weakening of the binding affinity of siRNA and dendrimer.

Chemoenzymatic Synthesis of Chiral Organosulfur Compounds

This thesis is primarily concerned with the enzyme- catalysed synthesis of sulfoxides using reductase and dioxygenase enzymes. Chapter 1 provides an introduction to the topic of redox chemistry with particular emphasis on the application of reductase and dioxygenase enzymes in organosulfur chemistry. Earlier literature methods for the production of enantiopure sulfoxides are reviewed. A brief discussion of the methods used for the determination of enantiomeric excess and absolute configuration is provided. Chapter 2 contains results obtained using a range of whole-cell bacteria each using a dimethyl sulfoxide reductase enzyme. The synthesis of a series of racemic sulfoxides and the development of appropriate CSPHPLC analytical methods is discussed. Kinetic resolutions of a series of sulfoxides have been achieved. Chapter 3 contains a presentation of results using dioxygenase enzymes as biocatalysts for the asymmetric sulfoxidation of dialkyl sulfoxides including thioacetal sulfoxides. A new range of monosulfoxides, cis-dihydrodiols and cis- dihydrodiol sulfoxides have been isolated in enantiopure form. Chapter 4 is focussed on the application of chiral sulfoxides in synthesis. A new chemoenzymatic route to diol sulfoxide enantiomers and the derived enantiopure phenols and catechols is discussed. The application of chemically synthesised sulfoxide enantiomers in the production of hydroxy sulfoxides is reported. Chapter 5 provides a full experimental section where the synthesis of sulfides and racemic sulfoxides is included. The methods used in the isolation and characterisation of bioproducts from the biotransformation are discussed and full experimental details given.

DNA Compaction by a Dendrimer

At physiological pH, a PAMAM dendrimer is positively charged and can effectively bind negatively charged DNA. Currently, there has been great interest in understanding this complexation reaction both for fundamental (as a model for complex biological reactions) as well as for practical (as a gene delivery material and probe for sensing DNA sequence) reasons. Here, we have studied the complexation between double-stranded DNA (dsDNA) and various generations of PAMAM dendrimers (G3-05) through atomistic molecular dynamics simulations in the presence of water and ions. We report the compaction of DNA on a nanosecond time scale. This is remarkable, given the fact that such a short DNA duplex with a length close to 13 nm is otherwise thought to be a rigid rod. Using several nanoseconds long MD simulations, we have observed various binding modes of dsDNA and dendrimers for various generations of PAMAM dendrimers at varying charge ratios, and it confirms some of the binding modes proposed earlier. The binding is driven by the electrostatic interaction, and the larger the dendrimer charge, the stronger the binding affinity. As DNA wraps/binds to the dendrimer, counterions originally condensed onto DNA (Na+) and the dendrimer (Cl-) get released. We calculate the entropy of counterions and show that there is gain in entropy due to counterion release during the complexation. MD simulations demonstrate that, when the charge ratio is greater than 1 (as in the case of the G5 dendrimer), the optimal wrapping of DNA is observed. Calculated binding energies of the complexation follow the trend G5 > 04 > 03, in accordance with the experimental data. For a lower-generation dendrimer, such as G3, and, to some extent, for G4 also, we see considerable deformation in the dendrimer structure due to their flexible nature. We have also calculated the various helicoidal parameters of DNA to study the effect of dendrimer binding on the structure of DNA. The B form of the DNA is well preserved in the complex, as is evident from various helical parameters, justifying the use of the PAMAM dendrimer as a suitable delivery vehicle.

Important role of the amino acid attached to tRNA in formylation and in initiation of protein synthesis in Escherichia coli

In attempts to convert an elongator tRNA to an initiator tRNA, we previously generated a mutant elongator methionine tRNA carrying an anticodon sequence change from CAU to CUA along with the two features important for activity of Escherichia coli initiator tRNA in initiation. This mutant tRNA (Mi:2 tRNA) was active in initiation in vivo but only when aminoacylated with methionine by overproduction of methionyl-tRNA synthetase. Here we show that the Mi:2 tRNA is normally aminoacylated in vivo with lysine and that the tRNA aminoacylated with lysine is a very poor substrate for formylation compared with the same tRNA aminoacylated with methionine. By introducing further changes at base pairs 4:69 and 5:68 in the acceptor stem of the Mi:2 tRNA to those found in the E. coli initiator tRNA, we show that change of the U4:A69 base pair to G4:C69 and overproduction of lysyl-tRNA synthetase and methionyl-tRNA transformylase results in partial formylation of the mutant tRNA and activity of the formyllysyl-tRNAs in initiation of protein synthesis. Thus, the G4:C69 base pair contributes toward formylation of the tRNA and protein synthesis in E. coli can be initiated with formyllysine. We also discuss the implications of these and other results on recognition of tRNAs by E. coli lysyl-tRNA synthetase and on competition in cells among aminoacyl-tRNA synthetases.