En la presente Tesis Doctoral se muestra el estudio de la proteína Ankyrin repeat and kinase domain containing 1 (ANKK1) en el linaje miogénico durante el desarrollo y en la edad adulta. El gen ANKK1 ha sido ampliamente relacionado con trastornos neuropsiquiátricos y endofenotipos dopaminérgicos en el cerebro. Sin embargo, la función de su proteína es todavía desconocida. La localización del gen ANKK1 en un clúster genómico conservado a lo largo de la evolución que podría estar implicado en neurogénesis, y la expresión de su proteína en progenitores neurales y su relación con el ciclo celular, han relacionado este gen con el neurodesarrollo. ANKK1 pertenece a la familia Receptor-Interacting Proteins (RIP), cuyos miembros participan en la diferenciación de diversos tejidos, incluyendo el muscular. La observación de ANKK1 en miotúbulos embrionarios murinos nos llevó a plantearnos la hipótesis de la posible participación de esta proteína en el origen, el desarrollo y la regeneración muscular. Nuestros resultados muestran que ANKK1 es una proteína que participa en la biología muscular. Se localiza en precursores miogénicos durante el desarrollo embrionario murino y en las células satélite del músculo adulto. Además, los estudios in vitro utilizando mioblastos murinos y humanos muestran un patrón específico de la dinámica de sus isoformas: las isoformas ANKK1 quinasa (ANKK1-k) y ANKK1 completa (ANKK1-fl) se expresan en mioblastos y células satélite (SCs) quiescentes, mientras que sólo ANKK1-fl está presente en miotúbulos y SCs activadas. El transporte núcleo-citoplasmático de ANKK1 en mioblastos durante la diferenciación temprana se bloquea mediante la adición de leptomicina B, lo que indica que su salida del núcleo está mediada por exportinas. En el músculo adulto ANKK1 se expresa en las fibras de contracción rápida tipo II de metabolismo glicolítico. La activación de la vía glicolítica en mioblastos murinos incrementa la expresión de Ankk1. Todo ello confirma la relación entre la expresión de ANKK1 y el metabolismo glicolítico y explica la localización específica de la proteína en fibras musculares de contracción rápida. También se ha investigado la localización de ANKK1 en músculos de pacientes con diferentes distrofias musculares. Los mioblastos de pacientes con Distrofia Muscular de Duchenne (DMD) presentan una expresión alterada de ANKK1. La disminución de ANKK1 nuclear en estos mioblastos se asocia con un estadio celular más indiferenciado, definido por el incremento de expresión de PAX7. Paralelamente, en biopsias procedentes de pacientes con diferentes distrofias musculares, la expresión de ANKK1 se asocia con poblaciones celulares regenerativas, es decir, SCs y fibras regenerativas. En cuanto al estudio de su función, se ha investigado la participación de ANKK1 en el ciclo celular. La sobreexpresión de las variantes polimórficas de ANKK1 (A1-A2) en células HeLa incrementa la velocidad de progresión del ciclo celular, mientras que la sobreexpresión de la isoforma catalíticamente inactiva (K51R) la disminuye. En todos los casos, el porcentaje de células que alcanza la mitosis está reducido. Todo esto indica que la expresión de ANKK1 afecta tanto a la progresión del ciclo celular como al número de células que completan el ciclo. Finalmente, hemos estudiado la actividad quinasa de ANKK1. En las condiciones estudiadas, no se ha detectado esta actividad in vitro. Sin embargo, dado que es una RIP quinasa y su dominio quinasa es homólogo al resto de los miembros de la familia RIP, no podemos descartar que ANKK1 presente dicha actividad. En resumen, este Tesis Doctoral muestra por primera vez la participación de la proteína ANKK1 en la biología muscular desde el desarrollo embrionario hasta el músculo del adulto. Sin duda, ANKK1 es una proteína candidata a ser estudiada como biomarcador de enfermedad muscular. / The present Doctoral Thesis shows the study of the Ankyrin repeat and kinase domain containing 1 (ANKK1) protein in the myogenic lineage during development and in adulthood. The ANKK1 gene has been widely related to neuropsychiatric disorders and dopaminergic endophenotypes in the brain. However, the function of its protein is still unknown. The location of ANKK1 gene in a genomic cluster conserved throughout the evolution thay may be involved in neurogenesis, and the expression of its protein in neural progenitors and its relationship with the cell cycle, have linked ANKK1 gene to neurodevelopment. ANKK1 belongs to the Receptor-Interacting Proteins family (RIP), whose members participate in the differentiation of several tissues, including muscle tissue. The finding of the location of ANKK1 in murine embryonic myotubes led us to consider the hypothesis of the possible participation of this protein in muscles origin, development and regeneration. Our results show that ANKK1 is a protein that participates in muscle biology. It is located in myogenic precursors during murine embryonic development and in adult muscle satellite cells. In addition, in vitro studies using murine and human myoblasts show a specific pattern of the dynamics of its isoforms: the isoforms ANKK1 kinase (ANKK1-k) and ANKK1 full-length (ANKK1-fl) are expressed in myoblasts and quiescent satellite cells (SCs), whereas only ANKK1-fl is present in myotubes and activated SCs. The nuclear-cytoplasmic shuttle of ANKK1 in myoblasts during early differentiation is blocked by the addition of leptomycin B, which indicates that its exit from the nucleus is mediated by exportins. In the adult muscle ANKK1 is expressed in the Fast-Twitch muscle fibers type II with glycolytic metabolism. The activation of the glycolytic pathway in murine myoblasts increases Ankk1 expression. All this confirms the relationship between the expression of ANKK1 and the glycolytic metabolism and explains the specific location of the protein in Fast-twitch muscle fibers. The location of ANKK1 in the muscles of patients with different muscular dystrophies has also been investigated. The myoblasts of patients with Duchenne Muscular Dystrophy (DMD) present an altered expression of ANKK1. The decrease in nuclear ANKK1 in these myoblasts is associated with a more undifferentiated cell stage, defined by the increase in the expression of PAX7. In parallel, in biopsies from patients with different muscular dystrophies, the expression of ANKK1 is associated with regenerative cell populations, that is to say, SCs and regenerating fibers. Regarding the study of its function, we have investigated the participation of ANKK1 in the cell cycle. The overexpression of the polymorphic variants of ANKK1 (A1-A2) in HeLa cells increases the rate of progression of the cell cycle, while overexpression of the catalytically inactive isoform (K51R) decreases it. In all cases, the percentage of cells that reach mitosis is reduced. All this indicates that the expression of ANKK1 affects both the progression of the cell cycle and the number of cells that complete the cycle. Finally, we have studied the kinase activity of ANKK1. Under the conditions studied, this activity has not been detected in vitro. However, given that it is a RIP kinase and its kinase domain is homologous to the rest of the members of the RIP family, we cannot rule out that ANKK1 does not present this activity. In summary, this Doctoral Thesis shows for the first time the participation of the ANKK1 protein in muscle biology from embryonic development to adult muscle. Thus, we propose ANKK1 as a candidate protein to be studied as a biomarker of muscular disease. / En la present tesi doctoral es mostra l'estudi de la proteïna Ankyrin repeat and kinase domain containing 1 (ANKK1) en el llinatge miogènic durant el desenvolupament i en l'edat adulta. El gen ANKK1 ha estat àmpliament relacionat amb trastorns neuropsiquiàtrics i endofenotips dopaminèrgics en el cervell. No obstant això, la funció de la seua proteïna és encara desconeguda. La localització del gen ANKK1 en un clúster genòmic conservat al llarg de l'evolució que podria estar implicat en neurogènesi, i l'expressió de la seua proteïna en progenitors neurals i la seua relació amb el cicle cel¿lular, han relacionat aquest gen amb el neurodesenvolupament. ANKK1 pertany a la família Receptor-interacting Proteins (RIP), els membres de la qual participen en la diferenciació de diversos teixits incloent el muscular. L'observació d'ANKK1 en miotúbuls embrionaris murins ens va portar a plantejar-nos la hipòtesi de la possible participació d'aquesta proteïna en l'origen, el desenvolupament i la regeneració muscular. Els nostres resultats mostren que ANKK1 és una proteïna que participa en la biologia muscular. Es localitza en precursors miogènics durant el desenvolupament embrionari murí i en les cèl¿lules satèl¿lit del múscul adult. A més, els estudis in vitro utilitzant mioblasts murins i humans mostren un patró específic de la dinàmica de les seues isoformes: les isoformes ANKK1 quinasa (ANKK1-k) i ANKK1 completa (ANKK1-fl) s'expressen en mioblasts i cèl¿lules satèl¿lit (SCs) quiescents, mentre que només ANKK1-fl està present en miotúbuls i SCs activades. El transport nucli-citoplasmàtic d'ANKK1 a mioblasts durant la diferenciació primerenca es bloqueja mitjançant l'addició de leptomicina B, el que indica que la seua eixida del nucli està mediada per exportines. En el múscul adult ANKK1 s'expressa en les fibres de contracció ràpida tipus II de metabolisme glicolític. L'activació de la via glicolítica en mioblasts murins incrementa l'expressió d'Ankk1. Tot això confirma la relació entre l'expressió d'ANKK1 i el metabolisme glicolític i explica la localització específica de la proteïna en fibres musculars de contracció ràpida. També s'ha investigat la localització d'ANKK1 en músculs de pacients amb diferents distròfies musculars. Els mioblasts de pacients amb distròfia muscular de Duchenne (DMD) presenten una expressió alterada d'ANKK1. La disminució d'ANKK1 nuclear en aquests mioblasts s'associa amb un estadi cel¿lular més indiferenciat, definit per l'increment d'expressió de PAX7. Paral¿lelament, en biòpsies procedents de pacients amb diferents distròfies musculars, l'expressió d'ANKK1 s'associa amb poblacions cel¿lulars regeneratives, és a dir, SCs i fibres regeneratives. En quant a l'estudi de la seua funció, s'ha investigat la participació d'ANKK1 en el cicle cel¿lular. La sobreexpressió de les variants polimòrfiques d'ANKK1 (A1-A2) en cèl¿lules HeLa incrementa la velocitat de progressió del cicle cel¿lular, mentre que la sobreexpressió de la isoforma catalíticament inactiva (K51R) la disminueix. En tots els casos, el percentatge de cèl¿lules que arriba a la mitosi està reduït. Tot això indica que l'expressió d'ANKK1 afecta tant la progressió del cicle cel¿lular com al nombre de cèl¿lules que completen el cicle. Finalment, hem estudiat l'activitat quinasa d'ANKK1. En les condicions estudiades, no s'ha detectat aquesta activitat in vitro. No obstant això, atès que és una RIP quinasa i el seu domini quinasa és homòleg a la resta dels membres de la família RIP, no podem descartar que ANKK1 presente aquesta activitat. En resum, aquesta tesi doctoral mostra per primera vegada la participació de la proteïna ANKK1 en la biologia muscular des del desenvolupament embrionari fins al múscul de l'adult. Sens dubte, ANKK1 és una proteïna candidata a ser estudiada com a biomarcador de malaltia muscular. / Rubio Solsona, E. (2018). ESTUDIO DE LA PROTEÍNA ANKK1 EN LA FIBRA MUSCULAR: IMPLICACIONES EN DISTROFIAS MUSCULARES [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/100850

Risky alcohol use increases the risk of certain crimes such as drunk driving, spousal abuse and fighting. Around 60% of an individual’s alcohol use is attributable to genetic influences, however little is known regarding the specific genes that are involved in increasing the risk of risky alcohol use. Recent theories posit that some genes are so called susceptibility genes, meaning that carriers of certain genes or alleles are more susceptible both to positive and negative environments. The aim of the present study was to examine main and interaction effects of a possible susceptibility gene (ANKK1, which in previous research has been found to be related to risky alcohol use), and victimization as a negative environmental factor and parent-child relationship as a positive factor. Data were drawn from the RESUME project, and in the present study, 1.800 participants were included (47% males; 53% females; mean age of 22.15 years). Results showed no statistically significant main or interaction effect for ANKK1, but a statistically significant main effect was found for victimization and parent-child relationship. In conclusion, future research should include a larger sample size and use participants diagnosed with alcohol dependency. In addition, the susceptibility properties of ANKKI needs to be further examined, as the results from the present study indicate that ANKK1 is not a susceptibility gene. / Riskfylld alkoholkonsumtion kan öka risken för att vissa brott begås, såsom rattfylleri, partnervåld och slagsmål. Forskning har visat att omkring 60% av en individs alkoholkonsumtion kan förklaras av genetik, men det finns lite forskning kring just vilka gener som ökar risken för alkoholism. Nya teorier tror att vissa gener är så kallade sårbarhetsgener, vilket innebära att individer som bär på dessa gener eller särskilda alleler är mer sårbara för både positiva och negativa miljöfaktorer. Syftet med den nuvarande studien var att undersöka huvud-och interaktionseffekter av en potentiell sårbarhetsgen (ANKK1, som tidigare forskning visat har en påverkan på riskfylld alkoholkonsumtion), med utsatthet som den negativa faktorn och barn-föräldrarelation som den positiva faktorn. Data i form av enkätsvar och DNA prov från RESUME studien användes för den nuvarande studien, där 1,800 deltagare inkluderades (47% män; 53% av kvinnor; medelålder = 22.15). Resultaten visade inte på någon statistiskt signifikant huvud-eller interaktionseffekt för ANKKI, men det fanns en statistiskt signifikant huvudeffekt för utsatthet och barn-föräldrarelation. Slutsatsens som dras är att framtida studier bör fokusera på att ha ett större urval och använda sig av deltagare som är diagnosticerade med alkoholmissbruk. Utöver det bör framtida forskning fortsätta undersöka om ANKK1 är en sårbarhetsgen, eftersom resultaten från denna studie visar att den inte är en sårbarhetsgen.

Introdução: A grande variabilidade individual em resposta a fármacos antitabagismo sugere que tratamentos específicos podem ser mais efetivos em determinados subgrupos de fumantes. No contexto de medicina personalizada, o principal objetivo do presente estudo foi avaliar se polimorfismos nos genes CHRNA4, CHRNB2, CYP2B6 e ANKK1 estão associados com a resposta às terapias de cessação tabágica em pacientes provenientes de um programa de assistência ao fumante. Métodos: Estudo de coorte com 483 pacientes fumantes que receberam tratamento farmacológico (vareniclina, vareniclina e bupropiona, bupropiona em monoterapia ou coadministrada com terapia de reposição nicotínica). O sucesso na cessação tabágica foi considerado para os pacientes que completaram 6 meses de abstinência contínua. O teste de Fagerström para a dependência à nicotina (FTND) e o escore de consumo situacional Issa foram utilizados para avaliar a dependência à nicotina. Os polimorfismos CHRNA4 (rs1044396 e rs2236196), CHRNB2 (rs2072660 e rs2072661) e ANKK1 (rs1800497) foram genotipados pela análise da curva de melting e os polimorfismos CYP2B6 *9 (rs3745274), *4 (rs2279343), *5 (rs3211371) foram genotipados por restrição enzimática. Resultados: Os pacientes com o genótipo CC para o polimorfismo CHRNA4 (rs10443196) obtiveram menor taxa de sucesso no tratamento com vareniclina (29,5%) em comparação com os portadores dos genótipos CT ou TT (50,9%) (P=0,007; n=167). Os genótipos CT ou TT foram associados com maior odds ratio para o sucesso (OR=1,67; IC 95%=1,10-2,53; P=0,02), em um modelo multivariado. Os pacientes com o genótipo AA para o polimorfismo CYP2B6 (rs2279343) obtiveram maior taxa de sucesso no tratamento com bupropiona (48,0%) em comparação com portadores dos genótipos AG ou GG (35,5%) (P=0,05; n=237). O genótipo AA foi associado com maior odds ratio para o sucesso no tratamento (OR=1,92; IC 95%=1,08-3,42; P=0,03), em um modelo multivariado. Não foram observadas diferenças significativas nos escores FTND e Issa com relação aos polimorfismos estudados. Conclusão: Os polimorfismos CHRNA4 (rs1044396) e CYP2B6 (rs2279343) estão associados com a cessação tabágica em indivíduos tratados com vareniclina e bupropiona, respectivamente. Sugere-se que estes polimorfismos influenciam a resposta farmacológica e podem ser importantes para o desenho de uma farmacoterapia individualizada / Background: The large individual variability in response to drugs for smoking cessation suggests that specific treatments can be more effective in particular subgroups of smokers. In the context of personalized medicine, the main aim of the present study was to evaluate whether the CHRNA4, CHRNB2, CYP2B6 and ANKK1 polymorphisms are associated with response to smoking cessation therapies in patients from a smoker assistance program. Methods: This cohort study enrolled 483 smoking patients patients who received pharmacological treatment (varenicline, varenicline plus bupropion, bupropion in monoterapy or plus nicotine replacement therapy). Smoking cessation success was considered for patients who completed 6 months of continuous abstinence. Fagerström test for nicotine dependence (FTND) and Issa situational smoking scores were analyzed for nicotine dependence. The CHRNA4 (rs1044396 and rs2236196), CHRNB2 (rs2072660 and rs2072661) and ANKK1 rs1800497 polymorphisms were genotyped by high resolution melting analysis and the CYP2B6 *9 (rs3745274), *4 (rs2279343) and *5 (rs3211371) were genotyped by restriction fragment lenght polymorphisms. Results: Patients with CHRNA4 rs1044396 CC genotype had lower success rate in treatment with varenicline (29.5%) compared with carriers of CT or TT genotypes (50.9%) (P=0.007, n=167). The CT or TT genotypes were associated with higher odds ratio for success (OR=1.67, 95%CI=1.10-2.53, P=0.02), in a multivariate model. Patients with CYP2B6 rs2279343 AA genotype had higher success rate in treatment with bupropion (48.0%) compared with carriers of AG or GG genotypes (35.5%) (P=0.05, n=237). The AA genotype was associated with higher odds ratio for success (OR=1.92, 95%CI=1.08-3.42, P=0.03), in a multivariate model. We did not observe significant differences in the FTND and Issa scores according to the studied polymorphisms. Conclusion: The CHRNA4 rs1044396 and CYP2B6 rs2279343 are associated with smoking cessation in individuals on varenicline and bupropion terapies, respectively. We suggest that these polymorphisms influence the pharmacological response of these drugs and it might be important in the design of individualized pharmacotherapy

Introdução: A grande variabilidade individual em resposta a fármacos antitabagismo sugere que tratamentos específicos podem ser mais efetivos em determinados subgrupos de fumantes. No contexto de medicina personalizada, o principal objetivo do presente estudo foi avaliar se polimorfismos nos genes CHRNA4, CHRNB2, CYP2B6 e ANKK1 estão associados com a resposta às terapias de cessação tabágica em pacientes provenientes de um programa de assistência ao fumante. Métodos: Estudo de coorte com 483 pacientes fumantes que receberam tratamento farmacológico (vareniclina, vareniclina e bupropiona, bupropiona em monoterapia ou coadministrada com terapia de reposição nicotínica). O sucesso na cessação tabágica foi considerado para os pacientes que completaram 6 meses de abstinência contínua. O teste de Fagerström para a dependência à nicotina (FTND) e o escore de consumo situacional Issa foram utilizados para avaliar a dependência à nicotina. Os polimorfismos CHRNA4 (rs1044396 e rs2236196), CHRNB2 (rs2072660 e rs2072661) e ANKK1 (rs1800497) foram genotipados pela análise da curva de melting e os polimorfismos CYP2B6 *9 (rs3745274), *4 (rs2279343), *5 (rs3211371) foram genotipados por restrição enzimática. Resultados: Os pacientes com o genótipo CC para o polimorfismo CHRNA4 (rs10443196) obtiveram menor taxa de sucesso no tratamento com vareniclina (29,5%) em comparação com os portadores dos genótipos CT ou TT (50,9%) (P=0,007; n=167). Os genótipos CT ou TT foram associados com maior odds ratio para o sucesso (OR=1,67; IC 95%=1,10-2,53; P=0,02), em um modelo multivariado. Os pacientes com o genótipo AA para o polimorfismo CYP2B6 (rs2279343) obtiveram maior taxa de sucesso no tratamento com bupropiona (48,0%) em comparação com portadores dos genótipos AG ou GG (35,5%) (P=0,05; n=237). O genótipo AA foi associado com maior odds ratio para o sucesso no tratamento (OR=1,92; IC 95%=1,08-3,42; P=0,03), em um modelo multivariado. Não foram observadas diferenças significativas nos escores FTND e Issa com relação aos polimorfismos estudados. Conclusão: Os polimorfismos CHRNA4 (rs1044396) e CYP2B6 (rs2279343) estão associados com a cessação tabágica em indivíduos tratados com vareniclina e bupropiona, respectivamente. Sugere-se que estes polimorfismos influenciam a resposta farmacológica e podem ser importantes para o desenho de uma farmacoterapia individualizada / Background: The large individual variability in response to drugs for smoking cessation suggests that specific treatments can be more effective in particular subgroups of smokers. In the context of personalized medicine, the main aim of the present study was to evaluate whether the CHRNA4, CHRNB2, CYP2B6 and ANKK1 polymorphisms are associated with response to smoking cessation therapies in patients from a smoker assistance program. Methods: This cohort study enrolled 483 smoking patients patients who received pharmacological treatment (varenicline, varenicline plus bupropion, bupropion in monoterapy or plus nicotine replacement therapy). Smoking cessation success was considered for patients who completed 6 months of continuous abstinence. Fagerström test for nicotine dependence (FTND) and Issa situational smoking scores were analyzed for nicotine dependence. The CHRNA4 (rs1044396 and rs2236196), CHRNB2 (rs2072660 and rs2072661) and ANKK1 rs1800497 polymorphisms were genotyped by high resolution melting analysis and the CYP2B6 *9 (rs3745274), *4 (rs2279343) and *5 (rs3211371) were genotyped by restriction fragment lenght polymorphisms. Results: Patients with CHRNA4 rs1044396 CC genotype had lower success rate in treatment with varenicline (29.5%) compared with carriers of CT or TT genotypes (50.9%) (P=0.007, n=167). The CT or TT genotypes were associated with higher odds ratio for success (OR=1.67, 95%CI=1.10-2.53, P=0.02), in a multivariate model. Patients with CYP2B6 rs2279343 AA genotype had higher success rate in treatment with bupropion (48.0%) compared with carriers of AG or GG genotypes (35.5%) (P=0.05, n=237). The AA genotype was associated with higher odds ratio for success (OR=1.92, 95%CI=1.08-3.42, P=0.03), in a multivariate model. We did not observe significant differences in the FTND and Issa scores according to the studied polymorphisms. Conclusion: The CHRNA4 rs1044396 and CYP2B6 rs2279343 are associated with smoking cessation in individuals on varenicline and bupropion terapies, respectively. We suggest that these polymorphisms influence the pharmacological response of these drugs and it might be important in the design of individualized pharmacotherapy

Reduction in dopamine receptor D2, has been associated with insufficient brain reward, food addiction, obesity, and type 2 diabetes (T2D). Our aim was to assess whether the genetic variability responsible for this reduction is associated with poor dietary compliance and life style habits in T2D patients. Genetic-analysis was done for 109 T2D individuals who completed a 24-week randomized clinical trial and were assigned to follow either a low-GI or a high-fibre diet. Polymorphisms of TaqIA and C957T were compared with physical and biochemical measures. Regardless of dietary treatments, individuals with the C957T-T allele and the TaqIA-A2 allele were significantly associated with blood pressure reduction. Carriers of the T allele significantly lowered their body mass index (BMI) over the 24-week trial. Our findings suggest that the presence of the TaqIA-A2 allele is associated with a decrease in blood pressure. The C957T-T allele was associated with decrease in pressure and body weight.

Reduction in dopamine receptor D2, has been associated with insufficient brain reward, food addiction, obesity, and type 2 diabetes (T2D). Our aim was to assess whether the genetic variability responsible for this reduction is associated with poor dietary compliance and life style habits in T2D patients. Genetic-analysis was done for 109 T2D individuals who completed a 24-week randomized clinical trial and were assigned to follow either a low-GI or a high-fibre diet. Polymorphisms of TaqIA and C957T were compared with physical and biochemical measures. Regardless of dietary treatments, individuals with the C957T-T allele and the TaqIA-A2 allele were significantly associated with blood pressure reduction. Carriers of the T allele significantly lowered their body mass index (BMI) over the 24-week trial. Our findings suggest that the presence of the TaqIA-A2 allele is associated with a decrease in blood pressure. The C957T-T allele was associated with decrease in pressure and body weight.