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Adenovirus; Terapia contra el cáncer; Vectores virales Adenovirus; Cancer therapy; Viral vectors Adenovirus; Teràpia contra el càncer; Vectors virals PolyPurine Reverse Hoogsteen Hairpins (PPRHs) are gene-silencing DNA-oligonucleotides developed in our laboratory that are formed by two antiparallel polypurine mirror repeat domains bound intramolecularly by Hoogsteen bonds. The aim of this work was to explore the feasibility of using viral vectors to deliver PPRHs as a gene therapy tool. After treatment with synthetic RNA, plasmid transfection, or viral infection targeting the survivin gene, viability was determined by the MTT assay, mRNA was determined by RT-qPCR, and protein levels were determined by Western blot. We showed that the RNA-PPRH induced a decrease in cell viability in a dose-dependent manner and an increase in apoptosis in PC-3 and HeLa cells. Both synthetic RNA-PPRH and RNA-PPRH intracellularly generated upon the transfection of a plasmid vector were able to reduce survivin mRNA and protein levels in PC-3 cells. An adenovirus type-5 vector encoding the PPRH against survivin was also able to decrease survivin mRNA and protein levels, leading to a reduction in HeLa cell viability. In this work, we demonstrated that PPRHs can also work as RNA species, either chemically synthesized, transcribed from a plasmid construct, or transcribed from viral vectors. Therefore, all these results are the proof of principle that viral vectors could be considered as a delivery system for PPRHs. This research was funded by grant RTI2018-093901-B-I00 from Plan Nacional de Investigación Científica (Spain). Group holding the Quality Mention from Generalitat de Catalunya 2017-SGR-94. EA is awarded with fellowships from Generalitat de Catalunya (FI).

[spa] ANTECENDENTES: La fibrilación auricular (FA) es la arritmia más común, afectando a un 1-2% de la población general. Aunque la FA se ha asociado con numerosas alteraciones electrofisiológicas, moleculares y estructurales, su tratamiento no es eficaz en todos los pacientes. Desde hace una década, diversos estudios de “genome wide association” han asociado varios single nucleotide polymorphisms (SNP)s en las regiones cromosómicas 4q25, 1q21 y 16q22, entre otras, a un mayor riesgo de presentar FA. No obstante, los mecanismos que están detrás de cada uno de ellos todavía son desconocidos. Por otro lado, varios estudios han demostrado que factores como tener enfermedad en la válvula mitral, la edad o la función del ventrículo izquierdo pueden afectar a la homeostasis intracelular del calcio y a la actividad eléctrica en miocitos auriculares humanos. Sin embargo, aunque hay estudios que demuestran que la epidemiología de la FA es diferente entre hombres y mujeres, sólo se ha analizado el efecto del sexo sobre la homeostasis intracelular del calcio en modelos animales. HIPÓTESIS: La asociación específica de los diferentes SNPs con mecanismos moleculares y consecuencias electrofisiológicas concretas podría ser utilizada como biomarcador para la prevención del desarrollo de FA, así como para el tratamiento personalizado de pacientes teniendo en cuenta el efecto del sexo. OBJETIVOS: • Profundizar en los mecanismos moleculares que alteran la homeostasis del calcio en los portadores de múltiples variantes de riesgo de la región 4q25. • Investigar el efecto que tienen las variantes de riesgo de la región 1q21 sobre la corriente SK3 y la actividad eléctrica, así como la influencia de la homeostasis del calcio sobre la corriente SK3 en miocitos auriculares humanos. • Analizar las posibles interacciones que se pueden dar entre las diferentes variantes de riesgo 4q25 y 1q21, y cómo afectarían estas a la función de los miocitos auriculares humanos. • Determinar la influencia del sexo en la homeostasis intracelular del calcio en pacientes con FA. MÉTODOS: Los experimentos se llevaron a cabo en miocitos auriculares humanos aislados. Se utilizó la técnica de patch-clamp (voltaje-clamp o current-clamp en función del protocolo) para medir la corriente de calcio (ICa) y sus características electrofisiológicas, así como las corrientes transitorias de entrada (ITI) o las despolarizaciones espontáneas de membrana. Para visualizar el calcio intracelular se utilizó microscopia confocal. La expresión de proteínas se determinó mediante la técnica de western blot y su distribución se analizó con marcaje inmunofluorescente RESULTADOS: El análisis del efecto de múltiples variantes de riesgo de 4q25 sobre la ICa y la actividad eléctrica en miocitos auriculares humanos, mostró que los pacientes portadores de la variante de riesgo en rs1448818 (la más próxima a PITX2) presentaban una reducción significativa de la ICa, mientras que los pacientes portadores de las variantes de riesgo en rs2200733, rs13143308 y rs10033464 (aproximadamente a 150 kb de PITX2) presentaban un incremento significativo en la frecuencia de ITI. Teniendo en cuenta estos hallazgos en conjunto, el efecto parece estar asociado a la localización en la que se encuentran las variantes de riesgo, sugiriéndose un efecto inhibidor en el extremo de 4q25 en la que se encuentra la variante de riesgo rs1448818, y un efecto estimulador en la región central donde se encuentran las variantes de riesgo rs2200733, rs13143308 y rs10033464. Por otro lado, el análisis del efecto de la alteración en la homeostasis del calcio y de las variantes de riesgo 1q21 sobre la corriente SK3 y la actividad eléctrica en miocitos auriculares humanos mostró que los pacientes con FA, los portadores de la variante de riesgo en rs1448818 de 4q25 y los portadores de la variante rs13376333 de 1q21 presentaban una reducción en la corriente SK sensible a apamina. Además, el bloqueo de los canales SK3 con apamina demostró que una disminución de la función de estos se traducía en un incremento de las despolarizaciones espontáneas de la membrana. Finalmente, aunque la FA se ha asociado con una disminución de la ICa y un aumento de la frecuencia de ITI, el análisis del efecto del sexo sobre la homeostasis intracelular del calcio en miocitos auriculares humanos demostró que la ICa sólo se vio reducida en hombres con FA; mientras que permitió observar una mayor incidencia de ITI y despolarizaciones espontáneas de la membrana únicamente en mujeres, exacerbándose en aquellas con FA. CONCLUSIONES: 1. Las variantes de riesgo de la región 4q25 asociadas a FA tienen un efecto diferencial sobre la regulación del calcio intracelular en función de su localización. • Los pacientes portadores de la variante de riesgo en rs1448818, que se encuentra en el extremo más próximo a PITX2, presentan una reducción de la ICa que favorecería un acortamiento del periodo refractario y la reentrada eléctrica. • Por otro lado, los pacientes portadores de las variantes de riesgo en rs2200733, rs13143308 o rs10033464, situadas a unas 150 kb de PITX2, presentan un incremento en la frecuencia de ITI que favorecería las despolarizaciones espontáneas de la membrana y actividad ectópica. Esto sugiere que la elección del tratamiento para prevenir o tratar la FA debería tener en cuenta las variantes de riesgo que presenta el paciente, ya que sería beneficioso restaurar la ICa en portadores de rs1448818 y reducir la liberación espontánea de calcio en portadores de rs13143308. 2. Las alteraciones en la homeostasis del calcio modifican la corriente SK sensible a apamina. • Así, la reducción de la ICa observada en pacientes con FA o en portadores de la variante de riesgo en rs1448818 reduce la corriente SK sensible a apamina. • Por otro lado, la variante de riesgo rs13376333 de la región1q21, ubicada en el intrón 1 del gen KCNN3, también reduce la corriente SK sensible a apamina. • Los efectos de las variantes de riesgo rs1448818 y rs13376333 no son complementarios. Estas reducciones de la corriente SK3 retrasarían la repolarización de las despolarizaciones espontáneas de membrana, facilitando la inducción de potenciales de acción espontáneos y arritmias inducidas por actividad ectópica. Por tanto, la restauración de la función de los canales SK3 podría ser una terapia farmacológica específica para la prevención o el tratamiento de los pacientes portadores de la variante rs13376333. 3. El sexo es un factor a tener en cuenta ya que tiene un efecto diferencial sobre la ICa y la frecuencia de ITI en miocitos auriculares de pacientes con FA. • La ICa sólo se ve reducida en hombres con FA. • La incidencia de ITI y despolarizaciones espontáneas de la membrana es mayor únicamente en mujeres. Estos resultados sugieren que las terapias dirigidas a reestablecer la ICa serían más efectivas en hombres con FA, mientras que las terapias dirigidas a reestablecer la actividad del RyR2 serían más efectivas en mujeres con FA., [eng] BACKGROUND: Atrial fibrillation (AF) is the most common arrhythmia, affecting 1-2% of the general population. Although AF has been associated with numerous electrophysiological, molecular and structural alterations, its treatment is not effective in all patients. For a decade, various genome wide association studies have associated several single nucleotide polymorphisms (SNP)s in the chromosomal regions 4q25, 1q21 and 16q22 with an increased risk of developing AF. Nevertheless, the mechanisms behind each of them are still unknown. On the other hand, several studies have shown that clinical factors such as mitral valve disease, age, or left ventricular function can affect intracellular calcium homeostasis and electrical activity in human atrial myocytes. However, although there are studies showing differences in the epidemiology of AF between men and women, the effect of sex on intracellular calcium homeostasis has only been analyzed in animal models. HYPOTHESIS: The specific association of the different SNPs with molecular mechanisms and specific electrophysiological responses could be used as biomarkers for the prevention of AF, as well as for personalized treatment of patients with AF, taking into account the influence of sex. AIMS OF THE THESIS: • Analyze the molecular mechanisms that alter calcium homeostasis in carriers of multiple risk variants of the 4q25 region. • Investigate the effects of 1q21 risk variants on SK3 current and electrical activity, as well as the influence of calcium homeostasis on SK3 current in human atrial myocytes. • Analyze the possible interactions that can occur between the different 4q25 and 1q21 risk variants, and their combined effect on electrophysiological function of human atrial myocytes. • Determine the influence of sex on intracellular calcium homeostasis in patients with AF. METHODS: Experiments were carried out on isolated human atrial myocytes. Patch-clamp technique (voltage-clamp or current-clamp depending on the protocol) was used to measure calcium current and its electrophysiological characteristics, as well as transient inward currents (ITI) or spontaneous membrane depolarizations. Confocal microscopy was used to visualize intracellular calcium. Protein expression was determined using the western blot technique and their distribution was analyzed with immunofluorescent marking. RESULTS: Analysis of the effect of all 4q25 risk variants on ICa and electrical activity in human atrial myocytes showed a significant reduction in ICa amplitude in patients carrying the risk variant in rs1448818 (the variant closest to PITX2), while patients carrying the risk variants in rs2200733, rs13143308 and rs10033464 (approximately located 150 kb upstream from PITX2) presented a significant increase in the ITI frequency. Taking these findings into account, the functional effect seems to be associated with the location of the risk variants, suggesting an inhibitory effect on ion channel activity in the region close to PITX2 where the risk variant rs1448818 is found, and a stimulatory effect in the area further away from PITX2 where the risk variants rs2200733, rs13143308 and rs10033464 are found. On the other hand, analysis of the effect of the alterations in calcium homeostasis and 1q21 risk variants on SK3 current and electrical activity in human atrial myocytes showed that patients with AF, carriers of the risk variant rs1448818 and carriers of the risk variant rs13376333 at 1q21 all exhibited a reduction in apamine-sensitive SK current. Furthermore, blocking the SK3 channels with apamin led to an increase in spontaneous membrane depolarizations. Finally, AF has generally been associated with a decrease in ICa and an increase in ITI frequency, but analysis of the effect of sex on intracellular calcium homeostasis in human atrial myocytes showed that ICa is only reduced in men with AF; while higher frequency of ITI and spontaneous membrane depolarizations is only observed in women and exacerbated in those with AF. CONCLUSIONS: 1. Risk variants in the 4q25 region associated with risk of AF have a differential effect on intracellular calcium regulation depending on their location. • Patients with risk variant at rs1448818, which is closest to PITX2, present a reduction in ICa that would favor a shortening of refractory period and electrical reentry. • On the other hand, patients with risk variants at rs2200733, rs13143308 or rs10033464, located approximately 150 kb upstream from PITX2, present an increase in the ITI frequency that favors spontaneous membrane depolarizations and ectopic activity. This demonstrates that the choice of treatment to prevent or treat AF should take into account the risk variants carried by the patient, suggesting that it would be beneficial to restore ICa in carriers of rs1448818 and to reduce spontaneous calcium release in carriers of rs13143308. 2. Altered calcium homeostasis and 1q21 variants modify the apamine-sensitive current. • The reduction in ICa observed in patients with AF or in carriers of the risk variant in rs1448818 reduces the apamine-sensitive SK current. • The risk variant rs13376333 in the 1q21 region, located in intron 1 of the KCNN3 gene, also reduce the apamine-sensitive current. • The effects of the risk variants rs1448818 and rs13376333 are not complementary. These reductions in SK3 current would delay the repolarization of spontaneous membrane depolarizations, facilitating the induction of spontaneous action potentials and arrhythmias induced by ectopic activity. Therefore, restoration of SK3 channel function might represent a specific therapy for the prevention or treatment of AF in carriers of the rs13376333 risk variant. 3. Sex is also a factor to take into account since it has a differential effect on ICa amplitude and ITI frequency in atrial myocytes of patients with AF. • ICa is only reduced in men with AF. • The incidence of ITI and spontaneous membrane depolarizations is higher in women only and exacerbated in those with AF. These results suggest that therapies aimed at reestablishing ICa would be more effective in men with AF, while therapies aimed at restoring RyR2 activity would be more effective in women with AF.

Some lung and ovarian tumors are connected to the loss of expression of SMARCA4 gene. In its promoter region, a 44-nucleotides long guanine sequence prone to form G-quadruplex structures has been studied by means of spectroscopic techniques (circular dichroism, molecular absorption and nuclear magnetic resonance), size exclusion chromatography and multivariate analysis. The results have shown that the central 21-nucleotides long sequence comprising four guanine tracts of disparate length is able to fold into a pH-dependent ensemble of G-quadruplex structures. Based on acid-base titrations and melting experiments of wild and mutated sequences, the formation of a C·C+ base pair between cytosine bases present at the two lateral loops is shown to promote a reduction in conformational heterogeneity, as well as an increase in thermal stability. The formation of this base pair is characterized by a pKa value of 7.1 ± 0.2 at 20 °C and 150 mM KCl. This value, higher than those usually found in i-motif structures, is related to the additional stability provided by guanine tetrads in the G-quadruplex. To our knowledge, this is the first thermodynamic description of this base pair in loops of antiparallel G-quadruplex structures. © 2019, The Author(s)., Gerard Pastor (University of Barcelona, Spain) and Esther Miralles (Centres Científics I Tecnològics, University of Barcelona, Spain) are thanked for their help in carrying out some of the experiments. Funding from Spanish government (CTQ2015-66254-C2-2-P and CTQ2017-84415-R), Italian government (PIANO DI SOSTEGNO ALLA RICERCA 2018 – Linea 2 azione B (DEFENS)) and recognition from the Autonomous Catalan government (2017SGR114) are acknowledged. The content is solely the responsibility of the authors. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

[cat] L’endometriosi és una malaltia ginecològica, associada a un component inflamatòri crònic, caracteritzada per la presència de teixit endometrial fora de l’úter. L’etiologia de la malaltia encara és manté incerta i el seu diagnòstic sovint presenta alts retrassos, degut essencialment a la manca de tests de diagnòstic no invasius. L’adenosina trifosfat (ATP), una molècula proinflamatoria, promou i afavoreix el manteniment de l’estat inflamatori de l’endometriosi. A més, l’ATP influeix directament en les principals manifestacions clíniques de la malaltia: el dolor i la infertilitat. La senyalització purinèrgica, el grup de respostes biològiques a nucleòtids extracel·lulars com l’ATP o nucleòsids com l’adenosina, es troba alterada en moltes malalties amb un important component inflamatori,com és l’endometriosi. Previament, el nostre grup ha demostrat l’expressió de les ectonucleotidases, els enzims que regulen els nivells extracel·lulars d’ATP i adenosina, en l’endometri no patològic, amb canvis dependents d’hormones al llarg del cicle menstrual. Per una altra banda, vam determinar un increment de l’activitat nucleotidasa en el fluid contingut en endometriomes ovàrics, una presentació freqüent d’endometriosi. La nostra hipòtesi és que la senyalització purinèrgica juga un paper en l’endometriosi i que l’expressió diferencial de les ectonucleotidases pot utilitzar-se com un nou biomarcador de la malaltia. L’objectiu principal d’aquesta tesi és conèixer el paper de la senyalització purinèrgica, en particular de les ectonucleotidases, en l’endometriosi, per tal d’augmentar el coneixement sobre la fisiopatologia d’aquesta malaltia i determinar-ne possibles biomarcadors d’us diagnòstic i/o terapèutic. En la present tesi, ens hem centrat en l’estudi de l’expressió de diferents ectoenzims en el fluid contingut en endometriomes mitjançant immunoassajos, i en el teixit endometrial eutòpic i ectòpic de dones amb endometriosi mitjançant immunohistoquímica i assajos d’activitat ectonucleotidasa in situ. A més, metodològicament, s’ha optimitzat un protocol per a la detecció simultània de l’expressió proteica i l’activitat nucleotidasa. Altrament, s’ha establert un mètode d’obtenció de cultius primaris d’estroma endometrial de dones amb i sense la patologia i s’ha validat com una eina útil d’estudi de l’endometriosi. Els resultats obtinguts en l’estudi del contingut dels endometriomes han determinat els ectoenzims nucleòtid pirofosfatasa/fosfodiesterasa 1 (NPP1) i l’adenosina desaminasa (ADA) com a candidats a biomarcadors per l’endometriosi. D’altra banda, l’assoliment d’una millora metodològica en l’estudi de les ectonucleotidases ens ha permès caracteritzar l’expressió de la nucleòsid trifosfat difosfohidrolasa 2 (NTPDasa2) en el teixit endometrial humà. Hem determinat l’NTPDasa2 com a un nou marcador de cèl·lules mare mesenquimals endometrials (eMSCs) SUSD2+ i com a marcador de l’estroma basal, troballes importants per la medicina regenerativa. En el teixit endometrial ectòpic de dones amb endometriosi profunda, el subtipus més greu, hem demostrat la pèrdua d’expressió de les ectonucleotidases CD39 i CD73, principal eix d’hidròlisi de l’ATP. Així mateix, s’ha identificat l’NPP3 com a un nou marcador histopatològic de la malaltia, ja que únicament s’expressa en l’estroma endometrial eutòpic i ectòpic de les pacients amb endometriosi. Així doncs, l’expressió diferencial de les ectonucleotidases pot emprar-se com a biomarcador de la malaltia i presenta la senyalització purinèrgica com una via d’interès pel disseny de noves estratègies diagnòstiques i terapèutiques per l’endometriosi.

Monogenic disorders are often the result of single point mutations in specific genes, leading to the production of non-functional proteins. Different blood disorders such as s-thalassemia, sickle cell disease, hereditary spherocytosis, Fanconi anemia, and Hemophilia A and B are usually caused by point mutations. Gene editing tools including TALENs, ZFNs, or CRISPR/Cas platforms have been developed to correct mutations responsible for different diseases. However, alternative molecular tools such as triplex-forming oligonucleotides and their derivatives (e.g., peptide nucleic acids), not relying on nuclease activity, have also demonstrated their ability to correct mutations in the DNA. Here, we review the Repair-PolyPurine Reverse Hoogsteen hairpins (PPRHs) technology, which can represent an alternative gene editing tool within this field. Repair-PPRHs are non-modified single-stranded DNA molecules formed by two polypurine mirror repeat sequences linked by a five-thymidine bridge, followed by an extended sequence at one end of the molecule which is homologous to the DNA sequence to be repaired but containing the corrected nucleotide. The two polypurine arms of the PPRH are bound by intramolecular reverse-Hoogsteen bonds between the purines, thus forming a hairpin structure. This hairpin core binds to polypyrimidine tracts located relatively near the target mutation in the dsDNA in a sequence-specific manner by Watson-Crick bonds, thus producing a triplex structure which stimulates recombination. This technology has been successfully employed to repair a collection of mutants of the dhfr and aprt genes within their endogenous loci in mammalian cells and could be suitable for the correction of mutations responsible for blood disorders.

We report the in vivo regulation of Inosine-5´-monophosphate dehydrogenase 1 (IMPDH1) in the retina. IMPDH1 catalyzes the rate-limiting step in the de novo synthesis of guanine nucleotides, impacting the cellular pools of GMP, GDP and GTP. Guanine nucleotide homeostasis is central to photoreceptor cells, where cGMP is the signal transducing molecule in the light response. Mutations in IMPDH1 lead to inherited blindness. We unveil a light-dependent phosphorylation of retinal IMPDH1 at Thr159/Ser160 in the Bateman domain that desensitizes the enzyme to allosteric inhibition by GDP/GTP. When exposed to bright light, living mice increase the rate of GTP and ATP synthesis in their retinas; concomitant with IMPDH1 aggregate formation at the outer segment layer. Inhibiting IMPDH activity in living mice delays rod mass recovery. We unveil a novel mechanism of regulation of IMPDH1 in vivo, important for understanding GTP homeostasis in the retina and the pathogenesis of adRP10 IMPDH1 mutations.

[eng] The perpetuation of life depends on the ability to reproduce. All known forms of life use DNA as genetic information storage. Ribonucleotide reduction is the process by which ribonucleotides (NTPs) are transformed into deoxyribonucleotides (dNTPs), thereby forming the building blocks for DNA synthesis and repair. This reaction is catalyzed by a family of sophisticated enzymes, the ribonucleotide reductases (RNRs). All RNRs are metalloproteins that use a common, free-radical-based catalytic mechanism. However, depending on the specific mechanisms they use for radical generation, the type of cofactor they require, and differences in the structure of their protein complexes, RNR are divided into three different classes, namely class I, class II and class III. RNR classes also present different relationships with oxygen: class I RNRs are oxygen-dependent, class II RNRs are oxygen-independent, and class III RNRs are oxygen-sensitive. While eukaryotic organisms use only class I RNR, bacteria can encode all RNR classes in any possible combinations, which provides them with a valuable tool for to adapt to different environmental conditions. Facultative anaerobic bacteria can thrive in the presence or absence of oxygen. Numerous species significant for their clinical relevance are facultative anaerobes, as many environments inside host bodies feature hypoxic or anoxic conditions; microaerobic or anaerobic environments are also found in biofilms in chronic infections. However, a facultative anaerobic lifestyle also implies an increased cost in regulation complexity. This effect extends to the ribonucleotide reductases network, which in facultative anaerobic pathogens must be thoroughly regulated to react to the different challenges imposed by different oxygenation conditions, changing growth speeds, host defense mechanisms, etc. This study is focused on facultative anaerobic pathogens and the strategies they use to modulate ribonucleotide reduction and balance it under different environmental stimuli, variable oxygenation conditions, and during infection or biofilm formation. We used two very well-studied species: Pseudomonas aeruginosa and Escherichia coli. First, in P. aeruginosa, an opportunistic pathogen well-known for its chronic pulmonary infections, we explore the effects of the AlgZR two-components system, one of the main regulatory elements responsible for biofilm formation and chronification, in the control of the RNR network. We then conducted a comprehensive characterization of the master regulator of ribonucleotide reductases NrdR, from its general role to its specific mechanism of action, in both E. coli and P. aeruginosa. We also explored the differential roles of RNR classes during biofilm formation and the function that the master regulators of anaerobic metabolism play in their control. Finally, we used a technique developed in this work, a continuous-culture method named AnaeroTrans, to characterize in detail the gradual adaptation E. coli and P. aeruginosa withstand during the aerobic-anaerobic transition and, isolating this effect from all other consequences of biofilm formation, explore the dynamic actions of RNR classes and anaerobic regulators in the microaerobic range. Overall, this works provides a comprehensive description of the different roles ribonucleotide reductases play in anaerobic facultative pathogens and the regulatory mechanisms that control them., [spa] La reducción de ribonucleótidos es el proceso por el que éstos se convierten en desoxirribonucleótidos (dNTPs), formando así los precursores para la síntesis y la reparación del ADN. Esta reacción es catalizada por las ribonucleótido reductasas (RNR). Aunque todas las RNR son metaloproteínas que comparten un mismo mecanismo basado en radicales libres, éstas pueden separarse en tres clases (clase I, clase II, clase III) dependiendo del mecanismo que utilizan para la síntesis del radical, el tipo de cofactores que requieren, o las diferencias estructurales que presentan. Los organismos eucarióticos utilizan exclusivamente RNR de clase I, pero las bacterias pueden codificar todas las clases en cualquier combinación posible. Las bacterias anaeróbicas facultativas pueden crecer en presencia o ausencia de oxígeno. Numerosas especies significativas por su importancia clínica son anaeróbicas facultativas. No obstante, también han de asumir un coste superior en cuando a la complejidad de su regulación genética. Este efecto se extiende a las ribonucleótido reductasas, que, en patógenos anaeróbicos facultativos, deben poder responder a distintas concentraciones de oxígeno, cambios en la velocidad de crecimiento, mecanismos de defensa del anfitrión, etc. Este trabajo se centra en los patógenos anaeróbicos facultativos y las estrategias que usan para regular y equilibrar la síntesis de ribonucleótidos bajo diferentes estímulos ambientales y condiciones variables de oxigenación, así como durante la infección y la formación de biofilm. En primer lugar, exploramos el efecto sobre la red de las RNR del sistema de dos componentes AlgZR de P. aeruginosa, un sistema regulatorio relacionado con la síntesis de alginato, la formación de biofilm y la cronificación. Posteriormente, realizamos una caracterización completa de NrdR, el regulador principal de las ribonucleótido reductasas, analizando tanto su papel general sobre la red de las RNR como su mecanismo molecular, tanto en P. aeruginosa como en E. coli. También analizamos el papel que desempeñan las distintas clases de RNR durante la formación de biofilm y la función de los reguladores anaeróbicos generales en su control. Finalmente, usamos una técnica de cultivo continuo desarrollada para este trabajo que denominamos AnaeroTrans para el estudio detallado de la transición aeróbica-anaeróbica en P. aeruginosa y E. coli.

[eng] Maintenance of balanced nucleotide pools is necessary for cellular homeostasis. The nucleotide content of the cell is determined by the uptake of nucleosides by specific transporters, the salvage pathway and de novo synthesis of purine and pyrimidine nucleotides. These pathways are highly regulated at different levels and likely to be controlled by mechanisms allowing some sort of coordination among them. In order to decipher possible functional links within this machinery, in this work we have explored the relationship between nucleoside transporters and other elements of nucleotide metabolism. We have studied the interaction and the possible functional link of the human concentrative nucleoside transporter 3 (CNT3) with ADK (adenosine kinase), SAMHD1 (sterile alpha-motif (SAM) and histidine-aspartate (HD) domain-containing protein 1) and QDPR (quinoid dihydropteridin reductase), all of them somehow related to nucleotide metabolism. Biochemical and functional validation of these interactions has evidenced the interconnection between CNT3 and the machinery implicated in nucleotide metabolism. Furthermore, nucleoside analogues are used in the treatment of cancer but they are often administered as pro-drugs. Their metabolic activation takes profit of different metabolic steps known to be implicated in nucleotide metabolism. Thus, changes in these pathways can be extremely relevant in determining drug activation and action in cancer therapy. Recently, combinations of the nucleoside analog Ara-C (cytarabine) and FMS-like tyrosine kinase (FLT3) inhibitors have been developed as first line treatments of acute myeloid leukemia (AML). We have observed that FLT3 positively correlates with enzymes implicated in Ara-C metabolism such as deoxycytidine kinase (dCK), 5’-nucleotidase cN-II and SAMHD1. We have also demonstrated a link among these elements associated with FLT3 inhibition which supports the possibility of coordinated regulation of different elements of the nucleotide metabolic network by this tyrosine kinase receptor. These observations also suggest that, to avoid chemoresistance, it is important to consider the schedule of administration of Ara-C and FLT3 inhibitors in the treatment of AML. Overall, this study provides novel evidence suggesting the existence of protein networks able to promote coordinate regulation of the machinery implicated in nucleotide metabolism.

Many oncogenes enhance nucleotide usage to increase ribosome content, DNA replication, and cell proliferation, but in parallel trigger p53 activation. Both the impaired ribosome biogenesis checkpoint (IRBC) and the DNA damage response (DDR) have been implicated in p53 activation following nucleotide depletion. However, it is difficult to reconcile the two checkpoints operating together, as the IRBC induces p21‐mediated G1 arrest, whereas the DDR requires that cells enter S phase. Gradual inhibition of inosine monophosphate dehydrogenase (IMPDH), an enzyme required for de novo GMP synthesis, reveals a hierarchical organization of these two checkpoints. We find that the IRBC is the primary nucleotide sensor, but increased IMPDH inhibition leads to p21 degradation, compromising IRBC‐mediated G1 arrest and allowing S phase entry and DDR activation. Disruption of the IRBC alone is sufficient to elicit the DDR, which is strongly enhanced by IMPDH inhibition, suggesting that the IRBC acts as a barrier against genomic instability., Hierarchical organization of p21‐mediated G1 arrest and S‐phase DNA damage response signaling ensures genomic stability upon increasing inhibition of nucleotide synthesis enzyme IMPDH.

Colorectal cancer (CRC) shows aggregation in some families but no alterations in the known hereditary CRC genes. We aimed to identify new candidate genes which are potentially involved in germline predisposition to familial CRC. An integrated analysis of germline and tumor whole-exome sequencing data was performed in 18 unrelated CRC families. Deleterious single nucleotide variants (SNV), short insertions and deletions (indels), copy number variants (CNVs) and loss of heterozygosity (LOH) were assessed as candidates for first germline or second somatic hits. Candidate tumor suppressor genes were selected when alterations were detected in both germline and somatic DNA, fulfilling Knudson&rsquo, s two-hit hypothesis. Somatic mutational profiling and signature analysis were also performed. A series of germline-somatic variant pairs were detected. In all cases, the first hit was presented as a rare SNV/indel, whereas the second hit was either a different SNV (3 genes) or LOH affecting the same gene (141 genes). BRCA2, BLM, ERCC2, RECQL, REV3L and RIF1 were among the most promising candidate genes for germline CRC predisposition. The identification of new candidate genes involved in familial CRC could be achieved by our integrated analysis. Further functional studies and replication in additional cohorts are required to confirm the selected candidates.

[eng] Nucleosides and nucleotides have a key role in cell physiology being implicated in crucial processes such as DNA and RNA synthesis, cell signaling and metabolic regulation. Purine and pyrimidine nucleotide balance is required for cell homeostasis, being imbalance and nucleotide depletion associated with metabolic dysregulation and cancer development. Understanding the molecular mechanisms behind these events appears to be a suitable approach to uncover novel drug anticancer targets and, eventually, improve therapy. De novo nucleotide biosynthesis is a highly energetic expensive process tightly regulated at different levels. PAICS (phosphoribosylaminoimidazole carboxylase / phosphoribosylaminoimidazole succinocarboxamide synthetase) gene encodes the bifunctional enzyme ADE2, which catalyzes steps 6 and 7 of de novo purine nucleotide biosynthesis. This enzyme has been reported to have an important role in carcinogenesis. Here we have analyzed how PAICS is regulated when nucleoside availability disturbances are induced using HT-29 cell lines. PAICS expression appears to be modulated in a challenging situation when the inhibitions of de novo pyrimidine nucleotide biosynthesis and salvage pathways were combined. Moreover when cells were cultured in a nucleoside depleted medium we observed a regulation of human Concentrative Nucleoside Transporters (hCNTs) expression. This type of modulation suggests that extracellular nucleoside availability and de novonucleotide biosynthesis are metabolically interconnected.

Treballs Finals de Grau de Química, Facultat de Química, Universitat de Barcelona, Any: 2017, Tutors: Alan C. Spivey, Fèlix Urpí, Phosphorothioate oligonucleotides have been deeply studied as antisense therapy drugs. Their stereoselective synthesis, however, still depends on the use of chiral auxiliaries or modifications on the individual nucleosides, which slow their synthesis and increase costs. A catalytic asymmetric synthesis of these oligonucleotides would therefore be of great interest. Herein the catalytic asymmetric synthesis of dinucleoside phosphorothioates is attempted. After failing to optimise the conditions for the coupling using 4-DMAP-N-oxides as nucleophilic catalysts, hydrogen bond donors were envisaged as potential catalysts. Through the use of bifunctional thioureas containing a tethered amine, adequate conditions for the coupling were found and diastereoselectivity could be measured. Although low conversions were obtained, moderate selectivities were achieved for the synthesis of these dinucleosides

[eng] Malaria is a parasitic disease caused by Plasmodium parasites and it is transmitted by female Anopheles mosquito. P. falciparum has a complex life cycle that includes important stages in two different hosts: a mosquito and a human. The transmission between the human and the mosquito host also involves the transition between asexual and sexual forms of the parasites. Glycobiology includes the study of carbohydrate metabolism and glycoconjugate (glycoprotein and glycolipid) structures. Protozoan parasites synthesize different glycoconjugates for protection and to respond to changes in the environment. Glycoconjugates coat the parasite surface with carbohydrates generally different from the host ones. They are crucial for parasite virulence and survival. Until very recently the only glycan structures described in P. falciparum were the GPI-anchors, however other glycan structures have been found in the past few years as the N-glycans or C-mannosylation. The glycome consists in the complete set of glycosylations that an organism or a cell produces at a certain time point, therefore the description of the parasite glycome may help to understand better the host- pathogen interactions in parasitic diseases. Sugar nucleotides are activated forms of monosaccharides that are the donors of glycosyltransferases to form glyconjugates. They can be synthesized by a de novo pathway that consists in the bioconversion of an existing sugar or sugar nucleotide; or by a salvage pathway that involves an activation and a further pyrophosphorylation. The identification and quantification of the sugar nucleotides present in malaria parasites may help to describe its glycosylation profile. The first paper presented in the thesis describes the identification and quantification of the sugar nucleotides present in the parasite, among which we found: UDP-Glc, UDP-Gal, UDP-GlcNAc, GDP-Man and GDP-Fuc. We also investigated the salvage pathways present in the parasite but we couldn’t elucidate the presence of a fucose salvage pathway. Plasmodium parasites conserve homolog genes for the de novo biosynthetic pathway of GDP-Fuc: GMD and FS. We were able to prove the in vitro activity of GMD and FS enzymes and to show that both enzymes are required for the synthesis of fucose. GMD and FS are expressed along the intraerythrocytic life cycle and both enzymes localize in the cytoplasm of the parasite, as well as other parasite enzymes related with carbohydrate metabolism. The expression of a putative O-fucosyltransferase (PoFUT2) present in the parasite genome, together with the uptake of GDP-Fuc by parasite extracts suggested the presence of a fucose containing glycan. In the second paper, we characterized the enzymes responsible for the synthesis of GDP-Fuc, GMD and FS. We disrupted both genes in the parasite and analyzed the sugar nucleotide content present in the parasite. After GMD and FS disruption, GDP-Fuc was still detected in the parasite and no evidence of salvage mechanism was found. We described indirect evidence of a fucose containing glycan that was abrogated after the disruption of GMD. The last work here presented (not yet published) tries to characterize the enzyme that is probably responsible for the transfer of fucose to the glycoconjugate. We disrupted, by double crossover recombination, PoFUT2 gene in the human and in the rodent malaria parasite. The disruption of PoFUT2 does not have any significant effect for the viability and growth of the parasite along the parasite cycle in the human and in the mosquito host. These works open the door to new research lines to find an alternative pathway for obtaining fucose or GDP-Fuc. Obtaining evidence of the glycosylation state of PoFUT2 mutants and the characterization of other possible glycosylation reaction present in the parasite are other research topics to investigate., [spa] La malaria está causada por el parásito Plasmodium y se transmite mediante hembras del mosquito Anopheles. La glicobiología es el estudio de los procesos relacionados con los carbohidratos y las estructuras glicoconjugadas que forman. Los parásitos sintetizan glicoconjugados o proteínas de unión a glicanos, y muchas veces se encargan de mediar las interacciones huésped-patógeno. Los azúcares nucleótidos son formas activadas de monosacáridos que son usados por glicosiltransferasas para formar glicoconjugados. La identificación y cuantificación de estos azúcares nucleótidos en el parásito de la malaria puede contribuir a la definición de su perfil de glicosilación. El primer trabajo presentado en la tesis permitió identificar los azúcares nucleótidos presentes en el parásito, así como la posible presencia de un glicoconjugado que contenga fucosa, sintetizado a partir de la actividad O-fucosiltransferasa de una proteína homóloga anotada en el genoma del parásito, PoFUT2. El siguiente trabajo permitió caracterizar las enzimas implicadas en la síntesis de la GDP-fucosa, GMD y FS. Este artículo nos permitió mostrar evidencias indirectas de la presencia del glicococonjugado que contiene fucosa. A partir de la disrupción de los genes de biosíntesis descubrimos que el contenido de GDP-fucosa en parásitos mutantes no variaba con respecto a los parásitos salvajes. Sin embargo, la síntesis del gliconjugado sí que se reducía. El tercer trabajo (sin publicar) se centra en la caracterización de la enzima encargada de transferir la fucosa al glicoconjugado. La disrupción de PoFUT2 no parece tener ningún efecto en la viabilidad y crecimiento del parásito a lo largo del ciclo de éste en el humano y en el mosquito. Estos trabajos abren la puerta a nuevas investigaciones para descubrir una vía alternativa de obtención de GDP-Fucosa. La obtención de evidencias del estado de glicosilación de los mutantes de PoFUT2 y la caracterización de otras posibles glicosilaciones son otros temas a investigar.

Importance The causal direction and magnitude of the association between telomere length and incidence of cancer and non-neoplastic diseases is uncertain owing to the susceptibility of observational studies to confounding and reverse causation. Objective To conduct a Mendelian randomization study, using germline genetic variants as instrumental variables, to appraise the causal relevance of telomere length for risk of cancer and non-neoplastic diseases. Data Sources Genomewide association studies (GWAS) published up to January 15, 2015. Study Selection GWAS of noncommunicable diseases that assayed germline genetic variation and did not select cohort or control participants on the basis of preexisting diseases. Of 163 GWAS of noncommunicable diseases identified, summary data from 103 were available. Data Extraction and Synthesis Summary association statistics for single nucleotide polymorphisms (SNPs) that are strongly associated with telomere length in the general population. Main Outcomes and Measures Odds ratios (ORs) and 95%confidence intervals (CIs) for disease per standard deviation (SD) higher telomere length due to germline genetic variation. Results Summary data were available for 35 cancers and 48 non-neoplastic diseases, corresponding to 420 081 cases (median cases, 2526 per disease) and 1 093 105 controls (median, 6789 per disease). Increased telomere length due to germline genetic variation was generally associated with increased risk for site-specific cancers. The strongest associations (ORs [95%CIs] per 1-SD change in genetically increased telomere length) were observed for glioma, 5.27 (3.15-8.81); serous low-malignant-potential ovarian cancer, 4.35 (2.39-7.94); lung adenocarcinoma, 3.19 (2.40-4.22); neuroblastoma, 2.98 (1.92-4.62); bladder cancer, 2.19 (1.32-3.66); melanoma, 1.87 (1.55-2.26); testicular cancer, 1.76 (1.02-3.04); kidney cancer, 1.55 (1.08-2.23); and endometrial cancer, 1.31 (1.07-1.61). Associations were stronger for rarer cancers and at tissue sites with lower rates of stem cell division. There was generally little evidence of association between genetically increased telomere length and risk of psychiatric, autoimmune, inflammatory, diabetic, and other non-neoplastic diseases, except for coronary heart disease (OR, 0.78 [95%CI, 0.67-0.90]), abdominal aortic aneurysm (OR, 0.63 [95%CI, 0.49-0.81]), celiac disease (OR, 0.42 [95%CI, 0.28-0.61]) and interstitial lung disease (OR, 0.09 [95%CI, 0.05-0.15]). Conclusions and Relevance It is likely that longer telomeres increase risk for several cancers but reduce risk for some non-neoplastic diseases, including cardiovascular diseases.

The obligate intracellular lifestyle of Plasmodium falciparum and the difficulties to obtain sufficient amounts of biological material has hampered the study of specific metabolic pathways in the malaria parasite. Thus, for example, the pools of sugar nucleotides required to fuel glycosylation reactions have never been studied in-depth in well-synchronized asexual parasites or in other stages of its life cycle. These metabolites are of critical importance, especially considering the renewed interest in the presence of N -, O - and other glycans in key parasite proteins. In this work, we adapted a liquid chromatography tandem mass spectrometry method based on the use of porous graphitic carbon (PGC) columns and MS-friendly solvents to quantify sugar nucleotides in the malaria parasite. We report on the thorough quantification of the pools of these metabolites throughout the intraerythrocytic cycle of P. falciparum The sensitivity of the method enabled for the first time the targeted analysis of these glycosylation precursors in gametocytes, the parasite sexual stages that are transmissible to the mosquito vector.

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/120717

Next-generation sequencing techniques and genome-wide association study analyses have provided a huge amount of data, thereby enabling the identification of DNA variations and mutations related to disease pathogenesis. New techniques and software tools have been developed to improve the accuracy and reliability of this identification. Most of these tools have been designed to discover and validate single nucleotide variants (SNVs). However, in addition to germ-line mutations, human tissues bear genomic mosaicism, which implies that somatic events are present only in low percentages of cells within a given tissue, thereby hindering the validation of these variations using standard genetic tools. Here we propose a new method to validate some of these somatic mutations. We combine a recently developed software with a method that cuts DNA by using restriction enzymes at the sites of the variation. The non-cleaved molecules, which bear the SNV, can then be amplified and sequenced using Sanger's technique. This procedure, which allows the detection of alternative alleles present in as few as 10 of cells, could be of value for the identification and validation of low frequency somatic events in a variety of tissues and diseases., Fundación BBVA (FBBVA) and the MICINN.We also acknowledge support from the Fundación Reina Sofía and grants from the Plan Nacional (MINECO) to JA (SAF-2014-53040-P) and ES (SAF-2013-4320).

Brain neurons offer diverse responses to stresses and detrimental factors during development and aging, and as a result of both neurodegenerative and neuropsychiatric disorders. This multiplicity of responses can be ascribed to the great diversity among neuronal populations. Here we have determined the metabolomic profile of three healthy adult human brain regions—entorhinal cortex, hippocampus, and frontal cortex—using mass spectrometry-based technologies. Our results show the existence of a lessened energy demand, mitochondrial stress, and lower one-carbon metabolism (particularly restricted to the methionine cycle) specifically in frontal cortex. These findings, along with the better antioxidant capacity and lower mTOR signaling also seen in frontal cortex, suggest that this brain region is especially resistant to stress compared to the entorhinal cortex and hippocampus, which are more vulnerable regions. Globally, our results show the presence of specific metabolomics adaptations in three mature, healthy human brain regions, confirming the existence of cross-regional differences in cell vulnerability in the human cerebral cortex. This research was funded by the Spanish Ministry of Economy and Competitiveness, Institute Carlos III (FIS grants PI14/00757 and PI14/00328), and the Autonomous Government of Catalonia (2014SGR69 and 2014SGR168) to IF and RP. This study was co-financed by FEDER funds from the European Union (‘A way to build Europe’). RC received predoctoral fellowships from the Autonomous Government of Catalonia. We thank T. Yohannan for editorial help.

Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in Europe and in North America. It is caused by the neoplastic transformation of a population of B-lymphocytes coexpressing CD5, CD19 and CD23. Although the etiology is not well known there is evidence of the existence of a genetical base. CLL is not only caused by the accumulation of B-lymphocytes due to a failure in the apoptosis. It is also caused by the active proliferation of the B-cells in response to the signs sent by the environment. Its clinical course is variable. One third of CLL patients are affected by an indolent form that does not require treatment. Another third of patients with leukemia will require iterative therapies and a small fraction patients develop Richter syndrome that reduces its overall survival to 5 to 8 months. This variability is due to the heterogeneity of the molecules that participate in the pathogenesis. One of these molecules is CD38. Some studies have demonstrated that the presence of this marker confers a worse prognostic, specially the presence of one polymorphism of CD38, rs6449182. Fluradabine, clyclophosphamide and rituximab (FCR) is considered the gold standard therapy for young patients under 65 years with CLL because achieves high percentage of minimal residual disease (MRD). But it has not been studied if in CLL patients with this polymorphism, FCR continues offering these good results. Objective: To analyze the relationship between the polymorphisms of CD38 and treatment outcomes in CLL patients treated with FCR. Methods: The design is a multicenter prospective cohort study. 338 patients will be recruited during 4 years. We will analyze the presence of the polymorphism and evaluate the minimal residual disease (MRD) after the treatment with FCR

Somatic mutations are the driving force of cancer genome evolution. The rate of somatic mutations appears to be greatly variable across the genome due to variations in chromatin organization, DNA accessibility and replication timing. However, other variables that may influence the mutation rate locally are unknown, such as a role for DNA-binding proteins, for example. Here we demonstrate that the rate of somatic mutations in melanomas is highly increased at active transcription factor binding sites and nucleosome embedded DNA, compared to their flanking regions. Using recently available excision-repair sequencing (XR-seq) data, we show that the higher mutation rate at these sites is caused by a decrease of the levels of nucleotide excision repair (NER) activity. Our work demonstrates that DNA-bound proteins interfere with the NER machinery, which results in an increased rate of DNA mutations at the protein binding sites. This finding has important implications for our understanding of mutational and DNA repair processes and in the identification of cancer driver mutations.

Purpose: To investigate new genetic risk factors and replicate reported associations with advanced age-related macular degeneration (AMD) in a prospective case–control study developed with a Spanish cohort. Methods: Three hundred and fifty-three unrelated patients with advanced AMD (225 with atrophic AMD, 57 with neovascular AMD, and 71 with mixed AMD) and 282 age-matched controls were included. Functional and tagging SNPs in 55 candidate genes were genotyped using the SNPlex TM genotyping system. Single SNP and haplotype association analysis were performed to determine possible genetic associations; interaction effects between SNPs were also investigated. Results: In agreement with previous reports, ARMS2 and CFH genes were strongly associated with AMD in the studied Spanish population. Moreover, both loci influenced risk independently giving support to different pathways implicated in AMD pathogenesis. No evidence for association of advanced AMD with other previous reported susceptibility genes, such as CST3, CX3CR1, FBLN5, HMCN1, PON1, SOD2, TLR4, VEGF and VLDLR, was detected. However, two additional genes appear to be candidate markers for the development of advanced AMD. A variant located at the 3¢ UTR of the FGF2 gene (rs6820411) was highly associated with atrophic AMD, and the functional SNP rs3112831 at ABCA4 showed a marginal association with the disease. Conclusion: We performed a large gene association study in advanced AMD in a Spanish population. Our findings show that CFH and ARMS2 genes seem to be the principal risk loci contributing independently to AMD in our cohort. We report new significant associations that could also influence the development of advanced AMD. These findings should be confirmed in further studies with larger cohorts.

BACKGROUND: The TP53 pathway, in which TP53 and its negative regulator MDM2 are the central elements, has an important role in carcinogenesis, particularly in BRCA1- and BRCA2-mediated carcinogenesis. A single nucleotide polymorphism (SNP) in the promoter region of MDM2 (309T > G, rs2279744) and a coding SNP of TP53 (Arg72Pro, rs1042522) have been shown to be of functional significance. METHODS: To investigate whether these SNPs modify breast cancer risk for BRCA1 and BRCA2 mutation carriers, we pooled genotype data on the TP53 Arg72Pro SNP in 7011 mutation carriers and on the MDM2 309T > G SNP in 2222 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). Data were analysed using a Cox proportional hazards model within a retrospective likelihood framework. RESULTS: No association was found between these SNPs and breast cancer risk for BRCA1 (TP53: per-allele hazard ratio (HR) = 1.01, 95% confidence interval (CI): 0.93-1.10, P(trend) = 0.77; MDM2: HR = 0.96, 95% CI: 0.84-1.09, P(trend) = 0.54) or for BRCA2 mutation carriers (TP53: HR = 0.99, 95% CI: 0.87-1.12, P(trend) = 0.83; MDM2: HR = 0.98, 95% CI: 0.80-1.21, P(trend) = 0.88). We also evaluated the potential combined effects of both SNPs on breast cancer risk, however, none of their combined genotypes showed any evidence of association. CONCLUSION: There was no evidence that TP53 Arg72Pro or MDM2 309T > G, either singly or in combination, influence breast cancer risk in BRCA1 or BRCA2 mutation carriers. British Journal of Cancer (2009) 101, 1456-1460. doi: 10.1038/sj.bjc.6605279 www.bjcancer.com Published online 25 August 2009 (C) 2009 Cancer Research UK

Sporadic Alzheimer disease (SAD) is the most prevalent neurodegenerative disorder. With the development of new generation DNA sequencing technologies, additional genetic risk factors have been described. Here we used various methods to process DNA sequencing data in order to gain further insight into this important disease. We have sequenced the exomes of brain samples from SAD patients and non-demented controls. Using either method, we found a higher number of single nucleotide variants (SNVs), from SAD patients, in genes present at the X chromosome. Using the most stringent method, we validated these variants by Sanger sequencing. Two of these gene variants, were found in loci related to the ubiquitin pathway (UBE2NL and ATXN3L), previously do not described as genetic risk factors for SAD., Fundación BBVA (FBBVA) and the MICINN; Fundación Reina Sofía and grants from the Plan Nacional (MINECO) to JA (SAF-2014-53040-P) and ES (SAF-2013-4320)

Inflammation is a complex process that implies the interaction between cells and molecular mediators, which, when not properly “tuned,” can lead to disease. When inflammation affects the eye, it can produce severe disorders affecting the superficial and internal parts of the visual organ. The nucleoside adenosine and nucleotides including adenine mononucleotides like ADP and ATP and dinucleotides such as P1,P4-diadenosine tetraphosphate (Ap4A), and P1,P5-diadenosine pentaphosphate (Ap5A) are present in different ocular locations and therefore they may contribute/modulate inflammatory processes. Adenosine receptors, in particularA2Aadenosine receptors, present anti-inflammatory action in acute and chronic retinal inflammation. Regarding the A3receptor, selective agonists like N6-(3-iodobenzyl)-5′-N-methylcarboxamidoadenosine (CF101) have been used for the treatment of inflammatory ophthalmic diseases such as dry eye and uveoretinitis. Sideways, diverse stimuli (sensory stimulation, large intraocular pressure increases) can produce a release of ATP from ocular sensory innervation or after injury to ocular tissues. Then, ATP will activate purinergic P2 receptors present in sensory nerve endings, the iris, the ciliary body, or other tissues surrounding the anterior chamber of the eye to produce uveitis/endophthalmitis. In summary, adenosine and nucleotides can activate receptors in ocular structures susceptible to suffer from inflammatory processes. This involvement suggests the possible use of purinergic agonists and antagonists as therapeutic targets for ocular inflammation.

[cat] La història evolutiva de les espècies queda impresa d’alguna manera en el seu genoma a nivell de les seqüències nucleotídiques. L’estudi dels nivells i patrons de la variació nucleotídica constitueix, per tant, una bona estratègia per inferir la importància relativa dels diferents factors evolutius que modelen aquesta variació. Drosophila subobscura és una espècie de dípter que pertany al grup obscura i que es caracteritza per presentar un elevat polimorfisme per inversions cromosòmiques. Així, aquesta espècie esdevé idònia per estudiar l’efecte d’aquests canvis estructurals del genoma a nivell de la variació nucleotídica. En la present tesi doctoral s’ha analitzat la variabilitat nucleotídica en múltiples regions no codificadores localitzades al llarg del cromosoma J en una població natural de D. subobscura de Barcelona. S’han seqüenciat aquestes regions en diferents línies homocariotípiques pel cromosoma J que es diferencien per la presència o absència d’una inversió relativament petita (J1) que és pràcticament l’única inversió que segrega a freqüències moderadament elevades en aquest cromosoma a la població estudiada. L’anàlisi de les regions no afectades per aquesta inversió ha permès inferir la història demogràfica recent de l’espècie en la regió Paleàrtica a través d’una anàlisi d’Approximate Bayesian Computation (ABC). El model demogràfic més probable entre els analitzats que pot descriure la variació genètica observada en l’actualitat en l’espècie és un model d’expansió poblacional, expansió ocorreguda fa aproximadament 110.000 anys. Altrament, l’anàlisi de la variació en les regions incloses dins la inversió J1 ha permès estudiar l’efecte de la inversió sobre la variabilitat nucleotídica. Els resultats obtinguts mostren una diferenciació genètica entre ordenacions altament significativa, i que és més elevada quan més pròximes es localitzen les regions a un punt de trencament. La disponibilitat del model demogràfic d’expansió ha permès, a més a més, inferir l’edat més probable de la inversió J1 utilitzant la metodologia d’ABC i un model demogràfic senzill el qual engloba no només la història evolutiva de la inversió J1, sinó també la història demogràfica recent de l’espècie. Els resultats indiquen que la inversió J1 és una inversió antiga amb una edat aproximada de 460.000 anys. Aquest estudi, junt amb estudis previs, mostra doncs que el polimorfisme per inversions de D. subobscura és bastant antic i provoca una forta estructuració de la variació nucleotídica en regions afectades per inversions. A més a més, l’estudi mostra que en aquesta espècie el patró de polimorfisme està caracteritzat per un excés de variants a baixa freqüència que és el resultat del procés d’expansió poblacional que va patir D. subobscura després del penúltim període glacial., [eng] A species evolutionary history is imprinted in some way in its genome nucleotide variation. The analysis of the levels and patterns of nucleotide variation in multiple regions constitutes a powerful approach to infer the relative importance of different evolutionary forces modulating this variation. Drosophila subobscura is characterized by a rich chromosomal inversion polymorphism, and it is, thus, a suitable species for studying the effect of this structural variation on levels and patterns of nucleotide variation. The main goal of this thesis project was to analyze nucleotide variation in multiple non-coding regions distributed along the J chromosome in a natural population of D. subobscura from Barcelona. The analysis of variation at regions not affected by inversions allowed inferring the species recent demographic history through an Approximate Bayesian Computation (ABC) analysis. The most likely demographic model that fits the data, among models considered, is characterized by an expansion that occurred approximately 110.000 years ago. In addition, the study of variation at regions affected by a relatively small inversion (J1) revealed a highly significant genetic differentiation between Jst and J1 arrangements. A simple demographic model including not only the evolutionary history of the inversion but also the recent demographic species history allows inferring that inversion J1 originated 460.000 years ago. This study, together with previous studies, shows that inversion polymorphism in D. subobscura is rather old and causes nucleotide variation to be highly structured in regions with inversions. The study also shows that the pattern of nucleotide variation is characterized in D. subobscura by an excess of low frequency variants resulting from the population expansion occurred in the species after the penultimate glacial period.

© 2014 Genre et al. Introduction: Rheumatoid arthritis is an inflammatory disease with high incidence of cardiovascular disease due to accelerated atherosclerosis. Osteoprotegerin (OPG) has been associated with increased risk of atherosclerotic disease in the general population. Several polymorphisms in the OPG gene with functional effects on cardiovascular disease in nonrheumatic individuals have been described. Therefore, we aimed to analyze the effect of three of these functional OPG polymorphisms on the risk of cardiovascular disease in a large and well-characterized cohort of Spanish patients with rheumatoid arthritis.

Animal olfactory systems have a critical role for the survival and reproduction of individuals. In insects, the odorant-binding proteins (OBPs) are encoded by a moderately sized gene family, and mediate the first steps of the olfactory processing. Most OBPs are organized in clusters of a few paralogs, which are conserved over time. Currently, the biological mechanism explaining the close physical proximity among OBPs is not yet established. Here, we conducted a comprehensive study aiming to gain insights into the mechanisms underlying the OBP genomic organization. We found that the OBP clusters are embedded within large conserved arrangements. These organizations also include other non-OBP genes, which often encode proteins integral to plasma membrane. Moreover, the conservation degree of such large clusters is related to the following: 1) the promoter architecture of the confined genes, 2) a characteristic transcriptional environment, and 3) the chromatin conformation of the chromosomal region. Our results suggest that chromatin domains may restrict the location of OBP genes to regions having the appropriate transcriptional environment, leading to the OBP cluster structure. However, the appropriate transcriptional environment for OBP and the other neighbor genes is not dominated by reduced levels of expression noise. Indeed, the stochastic fluctuations in the OBP transcript abundance may have a critical role in the combinatorial nature of the olfactory coding process.

Nucleoside transporters (NTs) mediate the uptake of nucleosides and nucleobases across the plasma membrane, mostly for salvage purposes. The canonical NTs belong to two gene families, SLC29 and SLC28. The former encode equilibrative nucleoside transporter proteins (ENTs), which mediate the facilitative diffusion of natural nucleosides with broad selectivity, whereas the latter encode concentrative nucleoside transporters (CNTs), which are sodium-coupled and show high affinity for substrates with variable selectivity. These proteins are expressed in most cell types, exhibiting apparent functional redundancy. This might indicate that CNTs play specific roles in the physiology of the cell beyond nucleoside salvage. Here, we addressed this possibility using adenoviral vectors to restore tumor cell expression of hCNT1 or a polymorphic variant (hCNT1S546P) lacking nucleoside translocation ability. We found that hCNT1 restoration in pancreatic cancer cells significantly altered cell-cycle progression and phosphorylation status of key signal-transducing kinases, promoted poly-(ADP ribose) polymerase hyperactivation and cell death, and reduced tumor growth and cell migration. Importantly, the translocation-defective transporter triggered these same effects on cell physiology. These data predict a novel and totally unexpected biological role for the nucleoside transporter protein hCNT1 that appears to be independent of its role as mediator of nucleoside uptake by cells, thereby suggesting a transceptor function. Cell Death & Disease Anastasis Stephanou Receiving Editor Cell Death & Disease 19th Apr 2013 Dr Perez-Torras Av/ Diagonal 643. Edif. Prevosti, Pl -1 Barcelona 08028 Spain RE: Manuscript CDDIS-13-0136R, 'CDDIS-13-0136R' Dear Dr Perez-Torras, It is a pleasure to inform you that your manuscript has been evaluated at the editorial level and has now been officially accepted for publication in Cell Death & Disease, pending you meet the following editorial requirements: 1) the list of the abbreviations is missing please include Could you send us the revised text as word file via e-mail and we will proceed and transfer the paper onto our typesetters. Please download, print, sign, and return the Licence to Publish Form using the link below. This must be returned via FAX to ++ 39 06 7259 6977 before your manuscript can be published

eld theory. The interactions determined from QCD are consistent with those extracted from hyperon-nucleon experimental data within uncertainties, and strengthen theoretical arguments that the strange quark is a crucial component of dense nuclear matter. The interactions between hyperons and nucleons are important for understanding the composition of dense nuclear matter. In high-density baryonic systems, the large values of the Fermi energies may make it energetically advantageous for some of the nucleons to transform into hyperons via the weak interactions, with the increase in rest mass being more than compensated for by the decrease in combined Fermi energy of the baryon-lepton system. This is speculated to occur in the interior of neutron stars, but a quantitative understanding of this phenomenon depends on knowledge of the hyperon-nucleon (YN) interactions in the medium. In this letter we use n scattering phase shifts in the 1 S0 and 3 S1 spinchannels calculated with Lattice QCD (LQCD) to quantify the energy shift of the hyperon in dense neutron matter, as might occur in the interior of a neutron star. Our results strongly suggest an important role for strangeness in such environments. Precise nucleon-nucleon (NN) interactions constrained by experiment and chiral symmetry, together with numerically small but important three-nucleon interactions, have served as input to rened many-body techniques for

Background: Recent data from neuroimaging, genetic and clinical trials and animal models suggest a role for altered glutamatergic neurotransmission in the pathogenesis of obsessive–compulsive disorder (OCD). The aim of this study was to investigate whether variants in the GRIN2B gene, the gene encoding the NR2 subunit of the N-methyl-d-aspartate (NMDA) glutamate receptor, may contribute to genetic susceptibility to OCD or to different OCD subphenotypes. Methods: Between 2003 and 2008, we performed a case–control association study in which we genotyped 10 tag single-nucleotide polymorphisms (SNPs) in the 3′ untranslated region (3′ UTR) of GRIN2B. We performed SNP association and haplotype analysis considering the OCD diagnosis and different OCD subphenotypes: early-onset OCD, comorbid tic disorders and OCD clinical symptom dimensions. Results: We enrolled 225 patients with OCD and 279 controls recruited from the OCD Clinic at Bellvitge Hospital (Barcelona, Spain). No significant difference in the distribution of alleles or genotypes was detected between patients with OCD and controls. Nonetheless, on analyzing OCD subphenotypes, the rs1805476 SNP in male patients (95% confidence interval [CI] 1.37–4.22, p = 0.002) and a 4-SNP haplotype in the whole sample (rs1805476, rs1805501, rs1805502 and rs1805477; odds ratio 1.92, 95% CI 1.22–3.01; permutation p = 0.023) were significantly associated with the presence of contamination obsessions and cleaning compulsions. Limitations: Study limitations included the risk of population stratification associated with the case–control design, use of psychiatrically unscreened blood donors as the control group, reduced sample size of participants with certain OCD subphenotypes and tested polymorphisms limited to 3′ UTR and exon 13 of GRIN2B. Conclusion: Our results converge with recent data suggesting a possible contribution of glutamatergic variants to the genetic vulnerability to OCD or at least to certain OCD manifestations. The dissection of OCD into more homogeneous subphenotypes may constitute a useful tool to disentangle the complex genetic basis of the disorder.

We unzip DNA molecules using optical tweezers and determine the sizes of the cooperatively unzipping and zipping regions separating consecutive metastable intermediates along the unzipping pathway. Sizes are found to be distributed following a power law, ranging from one base pair up to more than a hundred base pairs. We find that a large fraction of unzipping regions smaller than 10 bp are seldom detected because of the high compliance of the released single stranded DNA. We show how the compliance of a single nucleotide sets a limit value around 0.1 N/m for the stiffness of any local force probe aiming to discriminate one base pair at a time in DNA unzipping experiments., Main text: 4 pages, 3 figures. Supplementary Information: 18 pages, 15 figures

En el pescado, inmediatamente después de su muerte y durante su transporte a los puntos de comercialización, se suceden una serie de procesos autolíticos en el músculo, entre los que destacan la hidrólisis de los nucleótidos. Con el propósito de evaluar la influencia que ejerce la temperatura de almacenamiento sobre la concentración de nucleótidos para estimar la frescura del pescado, tres lotes de híbridos de cachama (Colossoma sp) se almacenaron en las siguientes condiciones: a) 0° C ± 3° C; b) 10° C ± 3° C y c) 27° C ± 3° C, por dos semanas. Las concentraciones de los nucleótidos individuales a lo largo del almacenamiento se determinaron por Cromatografía Líquida de Alta Resolución (HPLC) según el método de Iwamoto y col.[9], Los resultados indican que durante las primeras 24 horas las muestras almacenadas a 10° C presentaron la mayor tasa de hidrólisis del Adenosín Trifosfato (ATP), seguidas por las muestras almacenadas a 27° C y luego aquéllas almacenadas a 0° C. Las concentraciones de Inosina Monofosfato (IMP), Inosina (HxR) e Hipoxantina (HX) fueron independientes de la temperatura de almacenamiento. El incremento lineal en la concentración de HX observado en las tres temperaturas durante el período de almacenamiento, indica que éste puede ser usado como un buen índice de la pérdida de la frescura en estos híbridos. As soon as the fish dies and during its transportation to the retail centers, there are a series of autolytic processes in their tissues being the hydrolysis of the nucleotides the most important of them. These postmortem changes modify some of the original characteristics of the fish and determine its acceptance or rejection by the consumer. With the purpose of evaluating the influence that storage temperature exerts on the nucleotides concentration for estimating the freshness of fish, three lots of cachama’s hybrids (Colossoma sp) were kept under the following conditions: a) 0 °C ± 3° C; b) 10° C ± 3° C and c) 27° C ± 3° C, for two weeks at different temperatures. The concentrations of individual nucleotides through out the storage were performed using High Performance Liquid Chromatography (HPLC) by Iwamoto et al. [9] method. Results indicate that during the first 24 hours the samples stored at 10° C had the highest rate of Adenosin Monophosphate (ATP) hydrolysis followed by samples stored at 27°C and this in turn was higher than the one observed at 0° C. Inosine monophosphate (IMP), lnosine (HxR) and hipoxanthine (HX) concentrations were independent of storage temperature. The lineal increment of the HX concentration observed at all temperatures during the storage period indicates that it can be used as a good index of the loss of the freshness in these hybrids. 373 - 378 Bimestral

Tuberous sclerosis complex (TSC) is an autosomal dominant disease characterized by hamartoma formation in various organs. Two genes responsible for the disease, TSC1 and TSC2, have been identified. The TSC1 and TSC2 proteins, also called hamartin and tuberin, respectively, have been shown to regulate cell growth through inhibition of the mammalian target of rapamycin pathway. TSC1 is known to stabilize TSC2 by forming a complex with TSC2, which is a GTPase-activating protein for the Rheb small GTPase. We have identified HERC1 as a TSC2-interacting protein. HERC1 is a 532-kDa protein with an E3 ubiquitin ligase homology to E6AP carboxyl terminus (HECT) domain. We observed that the interaction of TSC1 with TSC2 appears to exclude TSC2 from interacting with HERC1. Disease mutations in TSC2, which result in its destabilization, allow binding to HERC1 in the presence of TSC1. Our study reveals a potential molecular mechanism of how TSC1 stabilizes TSC2 by excluding the HERC1 ubiquitin ligase from the TSC2 complex. Furthermore, these data reveal a possible biochemical basis of how certain disease mutations inactivate TSC2.

Se ha estudiado la evolución molecular de los genes del sistema olfatorio OS-E y OS-F en especies del subgénero Sophophora de Drosophila, concretamente en especies de los grupos melanogaster y obscura. Los genes OS (olfactory-specific) codifican para proteínas de la familia de las OBPs (Odorant Binding Proteins), proteínas de unión a odorantes (i/o feromonas), que debido a su función en el sistema de reconocimiento del medio externo podrían, a priori, haber sufrido cambios de tipo adaptativo. El objetivo ha sido estudiar el posible impacto de la selección natural positiva en la evolución de estos genes en Drosophila. Los resultados se han descrito en cuatro artículos:En el primer artículo se describe el análisis de la variabilidad nucleotídica en una región de unas 7kb que incluye los genes OS-E y OS-F en cuatro especies del subgrupo melanogaster de Drosophila, Drososphila melanogaster, D. simulans, D. mauritiana y D. erecta, así como en una población natural de Drosophila melanogaster. Los resultados indican que los genes OS-E y OS-F están presentes en las cuatro especies investigadas y mantienen su estructura génica. Las estimas de polimorfismo y divergencia nucleotídica soportan la hipótesis de que ambos genes son funcionales, aunque presentan diferencias en su tasa evolutiva y en su constricción funcional. Algunas de las observaciones no son compatibles con el modelo neutro.En el segundo artículo se describe la caracterización, secuenciación y análisis de la variabilidad nucleotídica de la región genómica que incluye los genes del sistema olfativo OS-E y OS-F en una población europea y en otra africana de la especie D. simulans. Las estimas de variación silenciosa y de la tasa de recombinación son mayores en D. simulans. que en D. melanogaster. Además, la población europea de D. simulans. presenta una estructura haplotípica inusual, donde la mitad de las secuencias son prácticamente idénticas (difiriendo en tan sólo una sustitución nucleotídica). Esta estructuración es incompatible con las predicciones de la teoría neutralista para poblaciones en equilibrio estacionario. En el tercer artículo se investiga la variación nucleotídica de la región que incluye los genes OS-E y OS-F en dos ordenaciones cromosómicas (O[3+4] y O3+4+23), presentes en dos poblaciones naturales de Drosophila subobscura. El análisis de los niveles y patrón del polimorfismo de DNA son consistentes con un origen monofilético de la inversiones. A pesar de que las dos ordenaciones cromosómicas están fuertemente diferenciadas, existen evidencias de que ha habido flujo genético entre las mismas, probablemente provocado por eventos de conversión génica. Los tests basados en la distribución de las frecuencias de las mutaciones indican que las ordenaciones cromosómicas no se encuentran en equilibrio. Bajo una hipótesis de expansión, se ha estimado que la inversión O23 se originó hace unos 0.25 millones de años.En el último artículo se analiza la evolución molecular de las secuencias codificadoras de los genes OS-E y OS-F en 14 especies del subgénero Sophophora de Drosophila. El trabajo se ha realizado combinando análisis por máxima verosimilitud de la variabilidad sinónima y no sinónima (mediante el uso de modelos evolutivos de codones), con la información de la putativa estructura tridimensional de la proteína. Se ha estimado que la duplicación que originó los dos genes pudo ocurrir hace unos 43-94 millones de años. Los resultados indican que a pesar de que ambos genes presentan una gran y similar constricción funcional, el gen OS-E presenta una tasa evolutiva global significativamente más alta. Los resultados de este capítulo, así como del trabajo en general, sugieren que la selección natural positiva actuó sobre posiciones concretas de la zona codificadora de estos dos genes, y pudo tener un papel importante en la preservación de los dos genes duplicados en el genoma., We have studied the molecular evolution (at intraspecific and interspecific levels) of two members of the OBP (Odorant Binding Protein) gene family (the OS-E and OS-F genes) in different Drosophila species. These genes likely originated by an old gene duplication event, still maintaining a high conservation at the gene structure, amino acid, and nucleotide levels. In this study, we detected significant differences in the evolutionary rate and functional constraint between the two genes. The results are unlikely for a neutral evolving region. We also found that levels of silent variation and recombination rates are significantly higher in "Drosophila simulans" than in "D. Melanogaster". Additionally, we detected that the European population of "D. Simulans" is highly structured, with a very unusual haplotype configuration that deviates significantly from the neutral expectations.In "D. Subobscura" this region maps on the O chromosome. In particular, the OS-region is located close to one of the breakpoints of the inversion O23. The analysis of the patterns of nucleotide variation at the OS-E and OS-F genes in "D. Subobscura" in two different chromosomal inversions showed that nucleotide variation is not at the steady-state equilibrium, but reflects the expansion process caused by the increase in frequency of the inversion. Finally, we analyzed nucleotide variation at the OS-E and OS-F genes in a number of Drosophila species to understand the evolutionary forces underlying their molecular evolution in this genus. We applied random and fixed-effects models, integrating information of DNA polymorphism, amino acid and nucleotide-based divergence, and 3D structure data to determine the selective constraint levels, and the putative role of positive selection in the evolutionary history of these genes, and especially in the maintenance of these duplicated gene copies. The results of these analyses allows concluding that positive selection is likely involved in the evolution of these genes in Drosophila.

Esta tesis recoge el estudio teórico de algunos de los más importantes tipos de interacciones débiles no covalentes en el ADN. Los sistemas de estudio van desde bases nitrogenadas y nucleósidos en fase gas hasta los ácidos nucleicos en solución. Concretamente se trataran las interacciones débiles pi-catión y los contactos entre bases con el objetivo de profundizar en su naturaleza. Dentro de las interacciones entre bases podemos diferenciar entre: (i) las interacciones de puente de hidrógeno impropio, (ii) las interacciones con bases modificadas, como la 8-aminoinosina y el difluorotolueno, y finalmente (iii) las interacciones no canónicas: Hoogsteen y Watson-Crick reverso, que pueden dar lugar a nuevas estructuras secundarias del ADN. Se utilizan todo un conjunto de técnicas teóricas que van desde la mecánica clásica hasta los más elevados niveles de mecánica cuántica, con la finalidad de aportar la máxima información estructural, reactividad y dinámica que complemente así los datos experimentales disponibles sobre las nucleobases y los ácidos nucleicos.En la primera parte de la presente tesis se describirán los métodos teóricos empleados para caracterizar las interacciones no covalentes. En la segunda parte se detallarán brevemente la estructura y las propiedades de los ácidos nucleicos. A continuación, se presentarán los resultados de las interacciones estudiadas. Y finalmente, se procederá a la discusión de los resultados obtenidos y a las conclusiones.

A novel two-base deletion in the 3-hydroxy-3-methylglutaryl coenzyme A lyase (HL) gene was found in a Spanish patient with homozygous 3-hydroxy-3-methylglutaric aciduria. Amplification by RT-PCR of the mRNAs showed that the gene was transcribed into three different mRNAs. One showed the complete deletion of exons 5 and 6 located between nucleotides 348 and 561 of the HL cDNA. The second transcript showed deletion of exon 6 only, and the third contained a two-base deletion CT in exon 6, corresponding to nucleotides 504 and 505 of the HL cDNA. These aberrant mRNAs are predicted to encode three abnormal HMG-CoA lyase proteins; the first (from skipped exons 5 and 6) lacks 71 amino acids, which represents 24% of the mature protein; the second, (from the skipping of exon 6, producing a frameshift) contains only 192 amino acids, the last 26 of which are missense amino acids preceding a stop codon; the third contains only 175 amino acids, the last 7 of which are missense. Northern blot analysis showed that the HL mRNA levels of the patient were 4% of the control. PCR quantitative analysis indicated that the mRNA lacking exons 5 and 6 was the most abundant, representing 88% of the total. The other two mRNAs represented 8% and 4%, respectively. In the genomic DNA only one CT deletion was found at positions +7 and +8 at beginning of exon 6. No mutations were observed in the splice donor, splice acceptor, or pyrimidine-rich sequences of the intronic regions flanking exons 5 and 6. All three aberrant mRNAs resulted only from the deletion of nucleotides CT. We suggest that this deletion may affect the interaction between the small nuclear ribonucleoproteins (snRNPs) and exon 6, and that, as a result, the abnormal splicing of the pre-mRNA produces two different aberrant transcripts. info:eu-repo/semantics/publishedVersion

A novel point mutation in the 3-hydroxy-3methyl-glutaryl coenzyme A lyase gene was found in a Turkish patient with homozygous 3-hydroxy-3-methylglutaric acidemia. Amplification by RT-PCR of the mRNA using a six different pairs of oligonucleotides produced no differences in four of the fragments amplified with respect to the control, but generated two fragments of different size. One was representative of a deletion of 126 bp and the other of an insertion of 78 bp. These abnormal mRNAs resulted from a G–>C transversion at the nucleotide +1 of an intron, which changed the invariant GT dinucleotide of the 5' donor splice site. This was associated with the occurrence of an alternative splicing, which led to the skipping of the whole exon of 126 bp, and also with the activation of one cryptic donor splice site in the same intron. These aberrant spliced mRNAs are predicted to encode two abnormal HMG-CoA lyase proteins: the first results in a protein with an internal deletion of 42 amino acids, whose enzyme activity is largely abolished, as the catalytic site was completely removed; the second contains 17 missense amino acids that precede a stop codon. Northern blot analysis showed that the overall content of these aberrantly spliced mRNAs in proband fibroblasts was the same as that found in control fibroblasts. However, hardly any transcript was observed corresponding to the inserted mutated mRNA when it was examined by a specific probe. To quantify the relative proportion of the two mRNAs, a quantitative RT-PCR (the DNA-mimic PCR reaction) was carried out. Results show that the proportion of the inserted mRNAs with respect to the deleted mRNA is only 1.2%. The father, mother, and two brothers of the proband were heterozygous in the G–>C mutation in the +1 nucleotide of the intron considered, while the two alleles of another brother were free of the mutation. info:eu-repo/semantics/publishedVersion

Fa aproximadament uns 20 anys del descobriment de la poli(ADP-ribosa), únic homopolímer que s'uneix de manera covalent a les proteïnes cromosòmiques. Des d'aleshores s'han fet detallats estudis de l'estructura, metabolisme, enzimologia, com també de les funcions fisiològiques relacionades amb el polímer. Nombroses evidències suggereixen que la poli(ADP-ribosa) es troba àmpliament distribuïda als eucariotes i està involucrada en la regulació de la proliferació cel·lular, síntesi de proteïnes i metabolisme dels àcids nucleics.Així mateix, també hi ha força dades que indiquen que la mono(ADP-ribosilació) és una manera comuna de modificar covalentment les proteïnes i la seva funció. L'ADP-ribosilació es l'única reacció bioquímica en la qual el NAD, un coenzim respiratori, és utilitzat com a donador de grups ADP-ribosil.La importància dels nucleòtids de piridina NAD i NADP com a coenzims de les reaccions d'oxidació-reducció cel·lulars pot observar-se fàcilment en considerar la utilització d'aquests compostos per les deshidrogenases i reductases que ocupen posicions crítiques de les vies metabòliques. El fet que el NAD sigui el més abundant dels coenzims ha suggerit que la funció d'aquest nucleòtid no es limita a les oxidacions biològiques. L'altre paper important del NAD és servir de substrat per a la modificació de les proteïnes per ADP-ribosilació.

[spa] Un tipo de proceso que ha sido ampliamente estudiado en bioinorgánica es la interacción de los iones metálicos 3D con los ácidos nucleicos y sus componentes. En el presente trabajo se ha procedido a la síntesis, caracterización (análisis elementales, espectroscopia infrarroja, análisis térmicos y calorimetría diferencial de barrido y espectroscopia visible) y estudio magnético y de espectroscopia Mossbauer de emisión de los complejos de CO2+ con los nucleótidos 2'AMP, 3'AMP, 5'AMP, 5'ATP, 5'GMP, 5'IMP, 5'CMP Y 5'UMP. Por tratamiento térmico de los complejos anteriores que presentan un entorno octaédrico se han obtenido los correspondientes complejos tetraédricos. De todos los estudios realizados se concluye que el Co(II), cuando interacciona con los nucleótidos, a excepción del 5'AMP y 5'CMP, presenta un entorno octaédrico con elevado grado de hidratación. Por tratamiento térmico se obtienen los compuestos en los que el Co(II) presenta un entorno tetraédrico. Los datos obtenidos de los estudios espectroscópicos y magnéticos confirman el cambio de coordinación y simetría característicos del Co(II).

Nucleo CMP Forte® is a nucleotide-based drug consisting of cytidinemonophosphate, uridinemonophosphate, uridin-ediphosphate and uridinetriphosphate. It has been prescribed for peripheral nervous system disorders, such as lum-bosciatalgia, diabetic or alcoholic polyneuropathy, or trigeminal neuralgia. Its effects on brain pathologies has re-ceived little attention. We examined its neuroprotective effects on cell toxicity induced by glutamate excitotoxicity or by 1-methyl-4-phenyl-pyridinium (MPP+), an in vitro cell model of Parkinson’s disease. We used the human dopaminergic cell line SH-SY5Y and a primary culture of rat cortical cells pre-treated with the drug for 24 hours and then exposed to MPP+ or glutamate at a range of concentrations. Cell viability was measured at different times. Nucleo CMP Forte® pre-treatment significantly increased the rate of cell division in SH-SY5Y cells, as well as the synthesis of triglycerides and phospholipids. More interestingly, drug pre-treatment significantly reduced MPP+- and glutamate-induced cell death in SH-SY5Y cells and in rat cortical cells. These results indicate that the nucleotides included in Nucleo CMP Forte® are promising therapeutic molecules for the prevention of neuronal death in brain caused by focal ischemia, Parkinson’s disease or other neurodegenerative pathologies. info:eu-repo/semantics/acceptedVersion