On the system of the functions x (s) / (s-r)k

"Vegeu el resum a l'inici del document del fitxer adjunt."

Síntesi de siRNAs (short interfering RNAs) modificats amb nucleobases 5-metil i 5-propinil pirimidíniques i avaluació de la seva activitat in vitro en cultius cel•lulars i de la seva estabilitat en sèrum humà. Disseny de siRNAs dirigits a la inhibició de l'expressió de mutacions de l'oncogen K-ras.

Projecte de recerca elaborat a partir d’una estada a la Stanford University, EEUU, entre 2007 i 2009. El present projecte es basa 1) en la síntesi de cadenes d'ARN dirigides a la inhibició de l'expressió gènica per un mecanisme d'ARN d'interferència (siRNAs o short interefering RNAs) i 2) en l'avaluació de l'activitat in vitro d'aquests oligonucleòtids en cultius cel•lulars. Concretament, la meva recerca ha estat enfocada principalment a l'estudi de cadenes de siRNA modificades amb nucleobases 5-metil i 5-propinil pirimidíniques. Es tractava d'avaluar l'efecte que exerceixen els factors estèrics en el major groove (solc major) dels siRNAs sobre la seva activitat biològica. En aquest sentit, he dut aterme síntesi de fosforamidits de nucleòsis pirimidínics modificats a la posició C-5 de la nucleobase. A continuació he incorporat aquestes unitats nucleosídiques en cadenes d'ARN emprant un sintetitzador d’ADN/ARN i he estudiat l'estabilitat dels corresponents dúplexs d'ARN mitjançant experiments de desnaturalització tèrmica. Finalment he dut a terme experiments d'inhibició de l'expressió gènica en cèl.lules HeLa per tal d'avaluar l'activitat biològia d'aquests siRNAs modificats. Els resultats d'aquests estudis han posat de manifest que la presència de grups voluminosos com el propinil a l'extrem 5' del dúplex de siRNA (definit per la cadena guia o antisense) influeix de forma molt negativa en la seva activitat biològica. En canvi, grups menys voluminosos com el metil hi influeixen positivament, de manera que algunes de les cadenes sintetitzades han resultat ser més actives que els corresponents siRNAs naturals (wild type siRNAs). A més, aquest tipus de modificació contribueix positivament en l'estabilitat de cadenes de siRNA en sèrum humà. Aquest treball ha estat publicat (Terrazas, M.; Kool, E.T. "Major Groove Modifications Improve siRNA Stability and Biological Activity" Nucleic Acids Res. 2009, in press).

Impaired fast-spiking, suppressed cortical inhibition and increased susceptibility to seizures in mice lacking Kv3.2 K+ channel proteins

Voltage-gated K+ channels of the Kv3 subfamily have unusual electrophysiological properties, including activation at very depolarized voltages (positive to −10 mV) and very fast deactivation rates, suggesting special roles in neuronal excitability. In the brain, Kv3 channels are prominently expressed in select neuronal populations, which include fast-spiking (FS) GABAergic interneurons of the neocortex, hippocampus, and caudate, as well as other high-frequency firing neurons. Although evidence points to a key role in high-frequency firing, a definitive understanding of the function of these channels has been hampered by a lack of selective pharmacological tools. We therefore generated mouse lines in which one of the Kv3 genes, Kv3.2, was disrupted by gene-targeting methods. Whole-cell electrophysiological recording showed that the ability to fire spikes at high frequencies was impaired in immunocytochemically identified FS interneurons of deep cortical layers (5-6) in which Kv3.2 proteins are normally prominent. No such impairment was found for FS neurons of superficial layers (2-4) in which Kv3.2 proteins are normally only weakly expressed. These data directly support the hypothesis that Kv3 channels are necessary for high-frequency firing. Moreover, we found that Kv3.2 −/− mice showed specific alterations in their cortical EEG patterns and an increased susceptibility to epileptic seizures consistent with an impairment of cortical inhibitory mechanisms. This implies that, rather than producing hyperexcitability of the inhibitory interneurons, Kv3.2 channel elimination suppresses their activity. These data suggest that normal cortical operations depend on the ability of inhibitory interneurons to generate high-frequency firing.

K(+) channel expression distinguishes subpopulations of parvalbumin- and somatostatin-containing neocortical interneurons

Kv3.1 and Kv3.2 K+ channel proteins form similar voltage-gated K+ channels with unusual properties, including fast activation at voltages positive to −10 mV and very fast deactivation rates. These properties are thought to facilitate sustained high-frequency firing. Kv3.1 subunits are specifically found in fast-spiking, parvalbumin (PV)-containing cortical interneurons, and recent studies have provided support for a crucial role in the generation of the fast-spiking phenotype. Kv3.2 mRNAs are also found in a small subset of neocortical neurons, although the distribution of these neurons is different. We raised antibodies directed against Kv3.2 proteins and used dual-labeling methods to identify the neocortical neurons expressing Kv3.2 proteins and to determine their subcellular localization. Kv3.2 proteins are prominently expressed in patches in somatic and proximal dendritic membrane as well as in axons and presynaptic terminals of GABAergic interneurons. Kv3.2 subunits are found in all PV-containing neurons in deep cortical layers where they probably form heteromultimeric channels with Kv3.1 subunits. In contrast, in superficial layer PV-positive neurons Kv3.2 immunoreactivity is low, but Kv3.1 is still prominently expressed. Because Kv3.1 and Kv3.2 channels are differentially modulated by protein kinases, these results raise the possibility that the fast-spiking properties of superficial- and deep-layer PV neurons are differentially regulated by neuromodulators. Interestingly, Kv3.2 but not Kv3.1 proteins are also prominent in a subset of seemingly non-fast-spiking, somatostatin- and calbindin-containing interneurons, suggesting that the Kv3.1–Kv3.2 current type can have functions other than facilitating high-frequency firing.

Copy number variation in the porcine genome inferred from a 60 k SNP BeadChip

Background: Recent studies in pigs have detected copy number variants (CNVs) using the Comparative Genomic Hybridization technique in arrays designed to cover specific porcine chromosomes. The goal of this study was to identify CNV regions (CNVRs) in swine species based on whole genome SNP genotyping chips. Results: We used predictions from three different programs (cnvPartition, PennCNV and GADA) to analyze data from the Porcine SNP60 BeadChip. A total of 49 CNVRs were identified in 55 animals from an Iberian x Landrace cross (IBMAP) according to three criteria: detected in at least two animals, contained three or more consecutive SNPs and recalled by at least two programs. Mendelian inheritance of CNVRs was confirmed in animals belonging to several generations of the IBMAP cross. Subsequently, a segregation analysis of these CNVRs was performed in 372 additional animals from the IBMAP cross and its distribution was studied in 133 unrelated pig samples from different geographical origins. Five out of seven analyzed CNVRs were validated by real time quantitative PCR, some of which coincide with well known examples of CNVs conserved across mammalian species. Conclusions: Our results illustrate the usefulness of Porcine SNP60 BeadChip to detect CNVRs and show that structural variants can not be neglected when studying the genetic variability in this species.