Realiza un análisis cromosómico y morfológico de Stenocercus ornatissimus, especie endémica perteneciente a la familia Tropiduridae; distribuido en las provincias de Canta, Huarochirí y Huaral en el departamento de Lima, a una altitud entre los 2000 msnm y 3400 msnm, actualmente categorizado como casi amenazado según la lista roja de la UICN “Unión Internacional de la Conservación de la Naturaleza”. Los estudios existentes en esta especie son descriptivos basándose en su morfología externa así como análisis filogenéticos, no abarcando estudios citogenéticos, en ninguno de las especies del género Stenocercus hasta el momento. Se busca observar diferencias morfológicas y citogenéticas en cuatro poblaciones alopátricas, distribuidos en los valles del Rímac, Chillón y Cañete, diferencias que estarían atribuidos a las barreras geográficas presentes por lo ríos de los mismos nombres. Los especímenes fueron colectados y procesados siguiendo las protocolos de Gonzáles et al. (2011), y Bickham (1975) previa estandarización, para el análisis citogenético, y siguiendo las claves de Torres-Carvajal (2007b) y Cadle (1998) para el análisis morfológico. Los resultados evidenciaron, en algunos casos, diferencias tanto a nivel citogenético como morfológico, observando la presencia de dos razas cromosómicas, una raza del Sur “2n=32” distribuida en el valle del río Rímac ubicado en la provincia de Huarochirí, y una raza Central 2n=34, en el valle del río Chillón en la provincia de Canta, no se observó diferencias morfológicas entre estas dos poblaciones. Mientras que, la población ubicada en el Valle del río Cañete ubicado en el límite sur de la distribución de la especie (provincia de Yauyos), mostró diferencias a nivel citogenético, con un xiv posible polimorfismo 2n=30-32, y diferencias a nivel morfológico, en el desarrollo y profundidad de los pliegues y bolsas del cuello, como la presencia de la bolsa de ácaros postfemoral, manteniéndose como una especie en confrontación “Stenocercus cf1 ornatissimus” frente a la especie en estudio, sugiriendo un posible proceso de especiación alopátrica basados en el modelo de poblaciones periféricas, el cual estaría atribuido a la barrera geográfica del valle Cañete. / Tesis

This thesis project is focused on the study of the signaling module formed by the NIMA-related protein Nek6, Nek7, and Nek9 and their function during early mitosis, with particular interest in centrosome separation and maturation. Nek9/Nercc1 was identified by Dr. Joan Roig. Nek9 is expressed in all cell lines and tissues studied is inactive during interphase while during mitosis is activated through phosphorylation by Plk1 which is in fact able to bind Nek9 and subsequently phosphorylates Nek9 on its activation loop. During mitosis Nek6 and Nek7 bind the C-terminal of Nek9. Once active, Nek9 can phosphorylate Nek6 and Nek7, thus activating them. Active Nek9 localizes at centrosome, suggesting that Nek9/Nek6-7 has important functions in the organization of microtubules during cell division. Confirming this idea, it has been shown that the microinjection of anti-Nek9 module induces arrest in prometaphase with disorganized spindle structures and misaligned chromosomes, or leads to abnormal mitosis resulting in aneuploidy. In the same direction, interference with the function of Nek7 or Nek6 leads to abnormal mitotic progression and spindle formation. We described how the Nek9/Nek6-7 module could provide a link connecting Plk1 and Eg5 in the context of centrosome separation. we analyzed the effects of Plk1, Eg5, Nek9, Nek6 or Nek7 down-regulation by RNAi on the extent of separation of duplicated centrosomes in prophase cells and we observed how this downregulation was affecting centrosome separation. We determine whether the activation of Nek9 or Nek6 could induce centrosome separation trasfecting cells with the active form of these two kinases; a considerable amount of cells that were in interphase shown separate centrosome demonstrating that Nek9/Nek6 are sufficient to induce centrosome separation. To test whether active Nek9 and Nek6 exerted their effect through the regulation of Eg5 we simultaneously transfected the cells with Eg5 siRNAs and we completely lost the centrosome separation described above. We demonstrated by immunofluorescence that the key event during centrosome separation was the recruitment of Eg5 at centrosomes and that the down-regulation of Plk1, Nek6, Nek7 or Nek9 resulted in prophase cells with unseparated centrosomes because Eg5 was not properly recruited. To prove whether the phosphorylation on Ser-1033 controls the accumulation of Eg5 to centrosomes and centrosome separation during early mitosis we transfected cells with wild type Eg5 or Eg5 S1033A; the wild type form of the kinesin was able to localize at centrosome and rescue the normal phenotype while Eg5 S1033A was not able to localize and resulted in cells delayed in mitosis. Plk1, the Nek9 activator, is involved in the regulation of centrosome maturation during early mitosis. Centrosome maturation refers to the process through which centrosomes increase size and microtubule nucleation activity and requires the accumulation of γ-TuRC complexes at centrosome. This recruitment depends on Nedd1 that acts as γ-Tubulin targeting factor. Plk1 depletion prevents accumulation of Nedd1 at centrosome. Our experiments show the importance of Nek9 in the regulation of centrosome maturation downstream of Plk1. Depletion of Nek9 by siRNA determined a decrease of γ-Tubulin and Nedd1 at centrosome. Further we investigated the upstream role of Plk1 depleting Plk1 and trasfecting active Nek9 and it was able to rescue the normal phenotype. Nek9 can interact with Nedd1 during mitosis and phosphorylates it provoking its accumulation at centrosome. The no-phosphorylable form of Nedd1 was not able to accumulate at centrosome and support the accumulation of γ-Tubulin there, determining a delay of the cells in prometaphase. Our results show that Nek9 is the link between Plk1 activity and the recruitment of Nedd1 to the centrosome and that the pathway formed by Plk1/Nek9/Nedd1 can be a key element in the control of mitotic centrosome maturation.

Publicación a texto completo no autorizada por el autor / Determina la influencia de la edad paterna en el desarrollo embrionario y euploidía, utilizando ciclos de Fecundación in vitro (FIV), con ovodonación y genética preimplantacional, por hibridación fluorescente in situ (FISH, de fluorescence in situ hybridization) o por hibridación genómica comparada (a-CGH, de array- comparative genomic hybridization). El presente estudio es de tipo analítico de cohortes retrospectivas. Se incluyeron 613 ciclos de ovodonación con genética preimplantacional, en la modalidad de screening (PGS). Se clasificó a la población en 2 cohortes, en función a la edad paterna: cohorte 1, no expuestos, <40 años (n= 231); cohorte 2, expuestos, ≥ de 40 años (n= 382). Los datos fueron recolectados de la base de datos computarizada de los dos Centros de FIV. Los resultados son en cuanto al desarrollo embrionario, la tasa de fecundación normal, resulta significativamente mayor en el grupo 1 (76.93%) que en el grupo 2 (73.96%) (p=0.0073). Asimismo, la tasa de falla de fecundación, es afectada significativamente por la edad paterna (p=0.028), resultando en 18.21% y 16.08% en los grupos 2 y grupo 1, respectivamente. La tasa de blastulación es significativamente menor en el grupo 2, 41.84% vs 45.78%, encontrado en varones de menor edad (p=0.0071). No se encontraron diferencias en la tasa de fecundación anormal y la calidad embrionaria en día 3, entre los grupos de estudio. Por otro lado, no se encontró diferencias en la tasa de euploidía embrionaria, determinada por FISH ni por aCGH, entre los dos grupos. Se concluye que el incremento de la edad paterna, afecta el desarrollo embrionario hasta blastocisto y la morfología, pero no la euploidía de los embriones. / Tesis

The main objectives of this work are:a) To test the predictions of suppressed-recombination chromosomal speciation models on two different lineages of mammals: rodents and rimates. Suppressed-recombination chromosomal speciation is still quite elusive as a mode of speciation in mammals. Experimental results are scarce and the first objective of this work is to analyze whole-genome data looking for traces of events of chromosomal speciation. Rodent and primate lineages were chosen for this search, not just because of their particular biological and cytological characteristics, which make them good candidates to have speciated by this mechanism, but also because they were the first mammalian organisms to be fully sequenced. b) To study the effects of chromosomal rearrangements on genic evolutionary rates. As have been seen in the introduction, there are many of potential interactions among chromosomal rearrangements and evolutionary rates, so the second goal of this work was to try to understand the impact of chromosomal rearrangements over substitution rates by means of other mechanisms not related with speciation. c) To distinguish individual contributions of different genomic factors in the potential association among chromosomal rearrangements and evolutionary rates.The third main goal of this thesis was to discern among the different factors that could be explaining the many associations between chromosomal and genic evolution that were detected in different studies.