Development of a sensitive multi-well colorimetric assay for active NFκB

The transcription factor nuclear factor κB (NFκB) is a key factor in the immune response triggered by a wide variety of molecules such as inflammatory cytokines, or some bacterial and viral products. This transcription factor represents a new target for the development of anti-inflammatory molecules, but this type of research is currently hampered by the lack of a convenient and rapid screening assay for NFκB activation. Indeed, NFκB DNA-binding capacity is traditionally estimated by radioactive gel shift assay. Here we propose a new DNA-binding assay based on the use of multi-well plates coated with a cold oligonucleotide containing the consensus binding site for NFκB. The presence of the DNA-bound transcription factor is then detected by anti-NFκB antibodies and revealed by colorimetry. This assay is easy to use, non-radioactive, highly reproducible, specific for NFκB, more sensitive than regular radioactive gel shift and very convenient for high throughput screening.

Branched co-polymers of histidine and lysine are efficient carriers of plasmids

We previously determined that a linear co-polymer of histidine and lysine (HK) in combination with liposomes enhanced the transfection efficiency of cationic liposomes. In the current study, we designed a series of HK polymers with increased branching and/or histidine/lysine ratio to determine if either variable affects transfection efficiency. In the presence of liposomes, the branched polymer with the highest number of histidines, HHK4b, was the most effective at enhancing gene expression. Furthermore, when serum was added to the medium during transfection, the combination of HHK4b and liposomes as a gene-delivery vehicle increased luciferase expression 400-fold compared to liposomes alone. In contrast to linear HK polymers, the higher branched HHK polymers were effective carriers of plasmids in the absence of liposomes. Without liposomes, the HHK4b carrier enhanced luciferase expression 15-fold in comparison with the lesser branched HHK2b carrier and increased expression by 5-logs in comparison with the HHK or HK carrier. The interplay of several parameters including increased condensation of DNA, buffering of acidic endosomes and differential binding affinities of polymer with DNA have a role in the enhancement of transfection by the HK polymers. In addition to suggesting that branched HK polymers are promising gene-delivery vehicles, this study provides a framework for the development of more efficient peptide-bond-based polymers of histidine and lysine.

Mechanisms underlying losses of heterozygosity in human colorectal cancers

Losses of heterozygosity are the most common molecular genetic alteration observed in human cancers. However, there have been few systematic studies to understand the mechanism(s) responsible for losses of heterozygosity in such tumors. Here we report a detailed investigation of the five chromosomes lost most frequently in human colorectal cancers. A total of 10,216 determinations were made with 88 microsatellite markers, revealing 245 chromosomal loss events. The mechanisms of loss were remarkably chromosome-specific. Some chromosomes displayed complete loss such as that predicted to result from mitotic nondisjunction. However, more than half of the losses were associated with losses of only part of a chromosome rather than a whole chromosome. Surprisingly, these losses were due largely to structural alterations rather than to mitotic recombination, break-induced replication, or gene conversion, suggesting novel mechanisms for the generation of much of the aneuploidy in this common tumor type.

Galactose-extended glycans of antibodies produced by transgenic plants

Plant-specific N-glycosylation can represent an important limitation for the use of recombinant glycoproteins of mammalian origin produced by transgenic plants. Comparison of plant and mammalian N-glycan biosynthesis indicates that β1,4-galactosyltransferase is the most important enzyme that is missing for conversion of typical plant N-glycans into mammalian-like N-glycans. Here, the stable expression of human β1,4-galactosyltransferase in tobacco plants is described. Proteins isolated from transgenic tobacco plants expressing the mammalian enzyme bear N-glycans, of which about 15% exhibit terminal β1,4-galactose residues in addition to the specific plant N-glycan epitopes. The results indicate that the human enzyme is fully functional and localizes correctly in the Golgi apparatus. Despite the fact that through the modified glycosylation machinery numerous proteins have acquired unusual N-glycans with terminal β1,4-galactose residues, no obvious changes in the physiology of the transgenic plants are observed, and the feature is inheritable. The crossing of a tobacco plant expressing human β1,4-galactosyltransferase with a plant expressing the heavy and light chains of a mouse antibody results in the expression of a plantibody that exhibits partially galactosylated N-glycans (30%), which is approximately as abundant as when the same antibody is produced by hybridoma cells. These results are a major step in the in planta engineering of the N-glycosylation of recombinant antibodies.

Defective regulatory volume decrease in human cystic fibrosis tracheal cells because of altered regulation of intermediate conductance Ca2+-dependent potassium channels

The cystic fibrosis transmembrane conductance regulator (CFTR) protein has the ability to function as both a chloride channel and a channel regulator. The loss of these functions explains many of the manifestations of the cystic fibrosis disease (CF), including lung and pancreatic failure, meconium ileus, and male infertility. CFTR has previously been implicated in the cell regulatory volume decrease (RVD) response after hypotonic shocks in murine small intestine crypts, an effect associated to the dysfunction of an unknown swelling-activated potassium conductance. In the present study, we investigated the RVD response in human tracheal CF epithelium and the nature of the volume-sensitive potassium channel affected. Neither the human tracheal cell line CFT1, expressing the mutant CFTR-ΔF508 gene, nor the isogenic vector control line CFT1-LC3, engineered to express the βgal gene, showed RVD. On the other hand, the cell line CFT1-LCFSN, engineered to express the wild-type CFTR gene, presented a full RVD. Patch-clamp studies of swelling-activated potassium currents in the three cell lines revealed that all of them possess a potassium current with the biophysical and pharmacological fingerprints of the intermediate conductance Ca2+-dependent potassium channel (IK, also known as KCNN4). However, only CFT1-LCFSN cells showed an increase in IK currents in response to hypotonic challenges. Although the identification of the molecular mechanism relating CFTR to the hIK channel remains to be solved, these data offer new evidence on the complex integration of CFTR in the cells where it is expressed.

Fluorescent light-induced chromatid breaks distinguish Alzheimer disease cells from normal cells in tissue culture.

The neurodegeneration and amyloid deposition of sporadic Alzheimer disease (AD) also occur in familial AD and in all trisomy-21 Down syndrome (DS) patients, suggesting a common pathogenetic mechanism. We investigated whether defective processing of damaged DNA might be that mechanism, as postulated for the neurodegeneration in xeroderma pigmentosum, a disease with defective repair not only of UV radiation-induced, but also of some oxygen free radical-induced, DNA lesions. We irradiated AD and DS skin fibroblasts or blood lymphocytes with fluorescent light, which is known to cause free radical-induced DNA damage. The cells were then treated with either beta-cytosine arabinoside (araC) or caffeine, and chromatid breaks were quantified. At least 28 of 31 normal donors and 10 of 11 donors with nonamyloid neurodegenerations gave normal test results. All 12 DS, 11 sporadic AD, and 16 familial AD patients tested had abnormal araC and caffeine tests, as did XP-A cells. In one of our four AD families, an abnormal caffeine test was found in all 10 afflicted individuals (including 3 asymptomatic when their skin biopsies were obtained) and in 8 of 11 offspring at a 50% risk for AD. Our tests could prove useful in predicting inheritance of familial AD and in supporting, or rendering unlikely, the diagnosis of sporadic AD in patients suspected of having the disease.

El granito como piedra de construcción en Madrid: durabilidad y puesta en valor

La tesis titulada “El granito como piedra de construcción en Madrid: durabilidad y puesta en valor” está estructurada en 6 artículos científicos publicados, 1 aceptado y otro en revisión, en diferentes revistas internacionales indexadas y un capítulo de un libro divulgativo. El uso de la piedra de construcción ha estado determinado por la proximidad de los recursos geológicos. Los materiales pétreos que históricamente se han utilizado en la Comunidad de Madrid provienen del Sistema Central Español y de la cuenca terciaria de Madrid. El sílex ha sido utilizado desde el siglo IX hasta el XI, cuando fue paulatinamente sustituido por la dolomía de Redueña y el granito, llamado tradicionalmente “piedra berroqueña” (Fort et al., 2013a, b). Los avances tecnológicos del siglo XVIII favorecieron la cantería subterránea de la caliza de Colmenar de Oreja. Sin embargo, la piedra berroqueña extraída a cielo abierto ha proporcionado la mayoría de la piedra de construcción utilizada en Madrid. La piedra berroqueña ha representado durante siglos la principal actividad económica en muchos pueblos de la Sierra de Guadarrama (Freire-Lista y Fort, 2015a, b). Estas sierras forman parte del Sistema Central Español. Se extienden en dirección suroeste-noreste a través de las provincias de Madrid, Segovia y Ávila. Ocupa un área aproximada de 100 km de largo por 40 km de ancho en donde existen diversas variedades de granito. Esta piedra ha dado origen a un elevado número de construcciones que suponen un importante pilar en el turismo histórico, cultural y rural del que disfruta actualmente la Comunidad de Madrid...

Calidad y durabilidad de áridos metamórficos empleados en capas de rodadura gallegas bajo el efecto de la sal y una climatología extrema

El uso del NaCl (sal común) en forma de salmuera está muy extendido durante el mantenimiento invernal de las carreteras españolas, pero este compuesto incide de forma negativa tanto en el medio ambiente como en las propias carreteras. Por este motivo, es necesario realizar un estudio detallado del efecto que tiene la sal cuando es esparcida sobre las carreteras en invierno. Además, esta sal puede permanecer en las superficies de las carreteras durante el verano, donde se pueden alcanzar temperaturas muy superiores a los 40º C. Por este motivo, es necesario considerar también estas condiciones en la evaluación de la durabilidad de una carretera. Por estos motivos, en esta tesis se ha estudiado la capa más superficial de una carretera y, por tanto, más expuesta a los efectos de la sal y agentes meteóricos (capa de rodadura). Estas capas están constituidas por mezclas asfálticas, cuyo mayor componente son los áridos de origen pétreo. Estos materiales son los principales responsables de la calidad y durabilidad de estas mezclas bituminosas por lo que es necesario analizar cómo se comportan bajo el efecto de la sal y en condiciones climatológicas adversas (invierno-verano). Los materiales estudiados fueron esquistos y anfibolitas explotadas en una cantera gallega, próxima a Santiago de Compostela. Esta cantera ha suministrado áridos y mezclas bituminosas a numerosas carreteras y autovías de la región, ya que dispone de su propia planta asfáltica...

An Arab Springboard for EU Foreign Policy? CEPS Paperbacks. January 2012

The EU has not been perceived as reacting very rapidly or effectively to the so-called Arab Spring. Events do appear to validate the idea underpinning the European Security Strategy (ESS) and the European Neighbourhood Policy (ENP): only where governments guarantee to their citizens security, prosperity, freedom and equality, can peace and stability last – otherwise, people will revolt. But in practice, in its southern neighbourhood the EU has acted in precisely the opposite manner, so the Arab Spring is occurring in spite of rather than thanks to EU policy. The ENP stands at a crossroads therefore: Can a new start be made? Which instruments and, in times of austerity, which means can the EU apply to consolidate democratization? And, finally, can the EU continue to wage an ENP without addressing the hard security dimension, especially as the US seem to be withdrawing from crisis management in the region – or shall it continue to leave that to others?

Educational attainment in the United States, March 1979 and 1978 /

Mode of access: Internet.

1978 University of Michigan Football Team

1979 University of Michigan Football Team

1980 University of Michigan Football Team

1992 University of Michigan Football Team

Teaching and testing for academic achievement : the role of language development /

"Spring 1991"--Cover.