Nuevas enzimas termoestables aplicadas a la síntesis de nucleósidos farmacológicamente activos

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 14-10-201

Dimensions of passion and their relationship to the risk of exercise addiction: Cultural and gender differences

This study was performed to investigate further the two-dimensional aspect of passion and its relationship to the risk of exercise addiction (REA) in nine nations and to clarify the unresolved gender differences. The here reported results stem from the reanalysis of data gathered in three previous empirical studies. The analyses demonstrated that harmonious (HP) and obsessive (OP) passion are two independent, non-interacting predictors of the REA, the prevalence of which was 12.1 % in the current sample that included 1448 people (age = 30.49 ± SD = 11.17 years; 55 % men), who exercised at least three hours per week. Furthermore, the results show that HP and OP could co-exist as a single ‘total’ or ‘true’ passion within the individual or in high-low HP and OP proportions, supporting the proposal for one, two-dimensional passion. Indeed, most people at REA demonstrated both high HP and high OP. The weekly amount of exercise was weakly associated with the two dimensions of passion. Relatively specific cultural differences in the REA and OP, but not HP, have emerged. The results also demonstrate that when a minimal weekly volume of training (i.e., 3 h) is a criterion for participant recruitment, no gender differences occur in the REA

Thermostable in vitro transcription-translation compatible with microfluidic droplets

Background: In vitro expression involves the utilization of the cellular transcription and translation machinery in an acellular context to produce one or more proteins of interest and has found widespread application in synthetic biology and in pharmaceutical biomanufacturing. Most in vitro expression systems available are active at moderate temperatures, but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures. Objectives: Develop an in vitro expression system operating at high temperatures compatible with enzymatic assays and with technologies that enable ultrahigh-throughput protein expression in reduced volumes, such as microfluidic water-in-oil (w/o) droplets. Results: We produced cell-free extracts from Thermus thermophilus for in vitro translation including thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable or superior to other thermostable in vitro expression systems, while the preparation procedure is much simpler and can be suited to different Thermus thermophilus strains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time. Conclusions: Cell-free extracts from Thermus thermophilus represent a simpler alternative to heavily optimized or pure component thermostable in vitro expression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughputThis work has received funding from the European Union\u2019s Research and Innovation Framework programs FP7 and Horizon 2020 under Grant Agreement numbers 324439, 635595, 685474, 695669 and 10100560 and from the Spanish Ministry of Economy and Competitiveness under grant number BIO-2013-44963-R. The CBM is funded by \u201CCentre of Excellence Severo Ochoa\u201D Grant CEX2021-001154-S from MICIU/AEI / https://doi.org/10.13039/501100011033 and receives institutional support by Fundaci\u00F3n Ram\u00F3n Arece

Thermus thermophilus nucleoside phosphorylases active in the synthesis of nucleoside analogues

Cells extracts from Thermus thermophilus HB27 express phosphorolytic activities on purines and pyrimidine nucleosides. Five putative encoding genes were cloned and expressed in Escherichia coli, and the corresponding recombinant proteins were purified and studied. Two of these showed phosphorolytic activities against purine nucleosides, and third one showed phosphorolytic activity against pyrimidine nucleosides in vitro, and the three were named TtPNPI, TtPNPII, and TtPyNP, respectively. The optimal temperature for the activity of the three enzymes was beyond the water boiling point and could not be measured accurately, whereas all of them exhibited a wide plateau of optimal pHs that ranged from 5.0 to 7.0. Analytical ultracentrifugation experiments revealed that TtPNPI was a homohexamer, TtPNPII was a monomer, and TtPyNP was a homodimer. Kinetic constants were determined for the phosphorolysis of the natural substrates of each enzyme. Reaction tests with nucleoside analogues revealed critical positions in the nucleoside for its recognition. Activities with synthetic nucleobase analogues, such as 5-iodouracil or 2,6-diaminopurine, and arabinosides were detected, supporting that these enzymes could be applied for the synthesis of new nucleoside analogs with pharmacological activities.Comunidad Autónoma de Madrid; Ministry of Science and Innovation (BIO2010-18875, CTQ2009-11801); Fundación Ramón ArecesPeer Reviewe

Thermus thermophilus Strains Active in Purine Nucleoside Synthesis

Several strains of Thermus thermophilus were tested in order to detect purine nucleoside synthase activity using pyrimidine nucleosides as the sugar-donor and adenine or hypoxanthine as bases. High productivity values (t =1 hr) were obtained while completely avoiding adenosine-deaminase degradation of the products. N-2-deoxy-ribosyltransferase activity is described for the first time in hyperthermophilic bacteria

Thermostable in vitro transcription-translation compatible with microfluidic droplets.

BACKGROUND: In vitro expression involves the utilization of the cellular transcription and translation machinery in an acellular context to produce one or more proteins of interest and has found widespread application in synthetic biology and in pharmaceutical biomanufacturing. Most in vitro expression systems available are active at moderate temperatures, but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures. OBJECTIVES: Develop an in vitro expression system operating at high temperatures compatible with enzymatic assays and with technologies that enable ultrahigh-throughput protein expression in reduced volumes, such as microfluidic water-in-oil (w/o) droplets. RESULTS: We produced cell-free extracts from Thermus thermophilus for in vitro translation including thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable or superior to other thermostable in vitro expression systems, while the preparation procedure is much simpler and can be suited to different Thermus thermophilus strains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time. CONCLUSIONS: Cell-free extracts from Thermus thermophilus represent a simpler alternative to heavily optimized or pure component thermostable in vitro expression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughput

Accurate time-frequency-wave number analysis to study coda waves

Geophysical Journal International, v. 158, n. 2, p. 577-591, 2004. http://dx.doi.org/10.1111/j.1365-246X.2004.02211.xInternational audienc