NUCLEIC ACIDS

Author: CASALS FALCÓ JOAN.
Year: 2004.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE QUÍMICA.
Place of preparation: FACULTAD DE QUÍMICA DE LA UNIVERSIDAD DE BARCELONA.

Summary: We have designed and synthesized conjugates acridina-oligonucleótido who can act as inhibitors of telomerase through the stability of structures cuádruplex of guaninas (G) formed at the ends teloméricos of human cells. The key intermediate in the synthetic route that has yielded the desired conjugates are derived acridínicos asymmetrical disustituidos, ocn carboxylic group at one end, which have become react 5'-amino-oligonucleótidos. The synthetic method developed has led to a small library of conjugated acridina-oligonucleótido introducing deviesidad level derivative acridínico, linker sequence oligonucleotídica and union between the two components. The conjugates obtained have been evaluated first, as stabilizers structures cuádruplex G formed by the human telomeric sequence through technical fret (fluorescence resonance energy transfer). Subsequently, it has assessed its ability to dela telomerase inhibition by testing trap (Telomeric repeat amplification protocol). Seha shown that conjugated obtained stabilize, but unless the acridinas to contain, cuádruplex G formed by the human telomeric sequence, and inhibiting telomerase addition, there has been an acceptable correlation between the results obtained in both experiments . On the other hand, there have been several synthesized oligonucleotides cícliros rich guaninas potentially formers cuádruplex of G and has been studied is structured techniques used ultraviolet (UV) spectroscopy, circular dichroism (CD) and nuclear magnetic resonance (NMR). In particular, it has been observed that dodecámero cyclical d greater Pttagggttaggg lesser forms a well defined structure of duádruplex bimolecular rate basket in the presence of sodium, finally, has studied the interaction of these oligonucleotides cyclical certain acridinas observed that stabilization structures of cuádruplex of Gy, in case you of oligonucleotide d greater Pttggttgg minor induce its structuring.

Author: TERRAZAS MARTÍNEZ MONTSERRAT.
Year: 2005.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAT DE QUÍMICA.
Place of preparation: FACULTAT DE QUÍMICA - UNIVERSITAT DE BARCELONA.

Summary: In this thesis has tried to develop new methodologies that allow changing the bases purínicas of nucleoside with two intentions clearly differentiated: on the one hand, marking with 15N, to facilitate structural analysis of nucleic acids; on the other hand, the search new species antiviral. In chapters 1 and 2 of this report has been studied reactivity of a wide range of 1-sulfonilinosinas with amines. The ultimate objective was to ascertain whether these substrates could see attacks on a nucleotide position electrófila C2 by amines to give rise to an intermediate open, as ciclación should carry inosinas modified in the N1. Of the various ways sulfonilación tested (N1-tosilo, N1-triisopropilbencenosulfonilo, N1-pentafluorobencenosulfonilo, N1-4-nitrobencenosulfonili, N1-2 ,4-dinitrobencenosulfonilo, N1 -2-nitrobencenosulfonilo, N1-mesilo and N1-triflilo), he gave better results in the reaction with aminíaco marking was for the group 2-nitrobencensulfonilo. Thus came with good performance 2'-desoxiinosinas fully marked on the N1. Moreover, the reaction with alkylamines, groups 2-nitrobencenosulfonilo and 2,4-dinitrobencenosulfonilo were the most suitable candidates for the position N1 of rent purine ring. This will also set up a new path toward 1-alquil-2'-desoxiinosinas. In the third chapter presents and discusses a new mechanism to transpose nucleobases purínicas allowing transform directly inosinas in adenosinas N1-alquiladas and [1-15N] adenosinas from 1-(2,4-dinitrobencenosulfonil) inosinas. In chapter 4, we have developed a new method of marking with 15N of the groups amino exocíclicos of purines. Our initial idea of introducing directly 15NH3 species purínicas activated so soft it has not been possible. Instead, the coupling catalysed Pd (0) - 6-bromopurinas and 2-bromoinosinas quantities estequiométricas [15N] benzamide was very effective. In addition, this protocol, together with development for the marking of N1 of 2'-desoxiinosinas, has enabled us to outline a new path to the [N-1- 15N2] adenosinas. Lastly, we have implemented procedures developed for the modification of nucleobases purínicas to the preparation of new species nucleosídicas directed to the inhibition of the above reverse transcriptase (RT) of the human immunodeficiency virus (HIV-1). Thus, in the fifth, we have focused on the design, synthesis and evaluation of the anti-HIV activity of species diméricas. This study has led to the discovery of a compound moderate inhibitory activity and low cytotoxicity.

Author: HERNANDEZ SANTOS DAVID.
Year: 2005.
University: OVIEDO [www.uniovi.es].
Place of defense: FACULTAD DE QUÍMICA.
Place of preparation: FACULTAD DE QUIMICA.

Summary: The methods for detecting DNA have undergone a major development over the past few years because of their potential to obtain valuable information about many genetic diseases, risks of environmental pollution or food processing, or, for example in forensic investigations. Of all the methods today are being developed to a large number of electrochemical sensor for the detection of DNA (usually called genosensores electrochemical), the high sensitivity of the transducer electrochemical coupled with the possibility of miniaturization of devices and microfabrication and the low cost of these systems makes them an innovative alternative for the detection of DNA. This paper describes the construction of new genosensores electrochemical small and single-use where detection is performed easily using DNA strands doubly marked, which makes comparisons between the physical and chemical properties of two detection systems and propose the most suitable conditions of use for each depending on the needs of the trial. Initially developed two strategies electrochemical detection of metal marks based on two reactions electroctalíticas (silver electroplating catalytic and catalytic reaction of hydrogen evolution), applying these methods to the detection of colloidal gold and various metal complexes, which might be AND used as a trademark, while evaluating the use of electrodes and carbon paste electrode serigrafiados carbon to carry out electrochemical detection and for subsequent use as transducers in building genosensores. Following the election of screen-printed electrode electrochemical as transducer, the catalytic reaction of hydrogen evolution as a method of detection and the use of the method of marking ULS (which uses a platinum complex to mark DNA strands, in this case with fluorescein) describing the construction of genosensores electrochemical where the sensor phase of ties through interaction estreptavidina-biotina using threads probe biotiniladas, and where it is possible to carry out detection cronoamperométrica direct platinum through the reaction of evolution catalytic hydrogen detection voltamperométrica or indirect enzyme of the florecerína using antibodies anti-fluoresceína marked with alkaline phosphatase and 3-indoxil phosphate as a substrate. The genosensores are developed using sequence-specific pathogen Streptococcus pneumoniae, and then apply to real samples through the detection of PCR products of gene TNFRSF21. The two models genosensor built devices are sensitive, reproducible and stable, capable of detecting a mutation in a sequence of bases used if the hybridization conditions adequate, although genosensores based on a detection enzyme are more reproducible, sensitive, based in detecting platinum allow analysis in less than half the time, greater ease and with the use of fewer reagents, with a consequent decrease in the cost per analysis.

Author: Roca Subirà Ignasi.
Year: 2006.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: Facultad de Ciencias.
Place of preparation: Facultad de Ciencias.

Summary: Ribonucleotide reductases (RNRs) are a group of enzymes that catalyze the reduction of ribonucleotides (NTPs) to deoxyribonucleotides (dNTPs), thus providing all organisms with optimal amounts of the necessary buildings blocks for DNA replication and repair. Three classes of RNRs have been characterized up to date according to their different mechanisms for radical generation, structural differences, allosteric regulation and oxygen dependence, all of them having in common the reaction mechanism and the use of an organic free radical to initiate catalysis. The genome of Streptococcus pyogenes (Group A Streptococcus [GAS]), a human pathogen responsible for many diverse infections, harbours the genes to synthesize two complete class Ib RNRs (nrdHEF/nrdI2 and nrdFIE* respectively), while most streptococci only bear the nrdHEF/nrdI2 genes. This work attempts to determine whether the presence of a second class Ib RNR in S. pyogenes accounts for a biological need or if it is simply the result of a vestigial burden. S. pyogenes is the first prokaryotic organism where two distinct and complete ribonucleotide reductases of the same class are studied. Linkage RT-PCR showed that both clusters are simultaneously transcribed and constitute two independent polycistronic units. The and subunits for these two systems were purified and biochemically characterized. The first system (nrdHEF) proved active and presented an EPR signal that matched that of other class Ib tyrosyl radicals. Activity was dependent on the addition of magnesium and DTT and required NDPs but not NTPs as a substrate. It was also stimulated by dATP as an allosteric effector but not by ATP, as expected in a class Ib enzyme. However, neither activity nor tyrosyl radical signal were ever detected from the nrdFIE* cluster. When aligned together with other class Ib enzymes, three out of the six iron binding residues within the subunit of the nrdFIE* cluster were found not to be conserved. The iron contents of the two NrdF proteins clearly showed that, contrary to the subunit from nrdHEF, the subunit from nrdFIE* was not capable of coordinating an iron centre in vitro under the conditions tested, which, in turn, impaired the generation of a tyrosyl radical. Heterologous complementation assays, however, showed that the nrdFIE* operon is fully functional in vivo when all three genes are present. In a similar manner, the nrdHEF system showed no complementation unless a functional NrdI protein was provided. Neither NrdI* nor NrdI2 from S. pyogenes rendered nrdHEF active, but it did addition of the NrdI2 protein form S. pneumoniae. The presence of an almost identical nrdFIE cluster in all Mycoplasma species leads us to suggest that the nrdFIE* operon was acquired by S. pyogenes through a lateral gene transfer event to compensate for the loss of a functional NrdI*. We have also studied the promoter regulating the transcriptional expression of the anaerobic ribonucleotide reductase of Escherichia coli (nrdDG). This promoter has been shown to be up-regulated by the pleiotropic transcriptional regulator FNR (fumarate and nitrate reduction), and two regions with significant similarity to the FNR consensus sequence were also found. In this work we have investigated the binding of FNR to the nrdDG promoter region and the effects of such binding on transcription. Gel retardation analysis with purified FNR* demonstrated FNR interaction at both sites, and studies with altered FNR boxes indicated that the upstream FNR-2 site is essential for the anaerobic activation of the nrdDG promoter. The FNR-1 site, however, is needed for the maximal expression of this promoter. Our results suggest that both sites act synergistically to coordinate expression in response to shifting oxygen concentrations.