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Trabajo presentado en el ciclo de conferencias CIC, celebrado en modalidad virtual el 03 de junio de 2021., The epigenome plays a crucial role on cell differentiation and response to different environmental and cellular conditions, including DNA damage. For that purpose, it can mediate important processes such as transcription or DNA damage response (DDR). Therefore, the modulation of different epigenetic marks must be a tightly controlled process, and its deregulation is involved in the onset of different diseases, including cancer. In this context, one of the most important mechanisms of epigenome regulation consists on regulating the activity of the epigenetic enzymes through post-translational modifications. For instance, the lysine acetyltransferase 5 (KAT5, also known as Tip60) is phosphorylated by different kinases in order to modulate the epigenetic pattern throughout the cell cycle. In addition, Tip60 also plays crucial roles during DDR, including the acetylation of the lysine 16 of histone 4 (H4K16), essential for chromatin decompaction, or acetylation of ATM. To our knowledge, nonetheless, any mechanism of Tip60 modulation and activation in the context of DNA damage has been described.

Tesis doctoral.-- Universidad de Salamanca, El ADN (ácido desoxirribonucleico) es la biomolécula constituyente del material genético, responsable de posibilitar todos los procesos biológicos de la célula y de transmitir dicha información genética a la descendencia (Avery et al., 1944). Teniendo en cuenta esto, se puede entender el genoma como la totalidad del material genético asociado al ADN que posee una célula, incluyendo no sólo las regiones codificantes (que, por ejemplo, en el caso del ser humano -tabla 1- constituyen únicamente el 1-1,5% del genoma), sino también aquellas regiones no codificantes. Entre estas últimas se incluyen los intrones (25% del genoma), secuencias de ADN repetitivo y duplicado (60%) y pseudogenes (14-13,5%), siendo todas ellas fundamentales para regular la expresión diferencial de las regiones codificantes de acuerdo con las necesidades celulares (Makalowski, 2001). Además, en el caso de células eucariotas, el genoma incluye tanto el ADN nuclear como el ADN contenido en diferentes orgánulos celulares de origen procariota, como mitocondrias o plastos.

Dynamic remodeling of chromatin requires acetylation and methylation of histones, frequently affecting the same lysine residue. These alternative epigenetic modifications require the coordination of enzymes, writers and erasers, mediating them such as acetylases and deacetylases. In cells in G0/G1, DNA damage induced by doxorubicin causes an increase in histone H4K16ac, a marker of chromatin relaxation. In this context, we studied the role that VRK1, a chromatin kinase activated by DNA damage, plays in this early step. VRK1 depletion or MG149, a Tip60/KAT5 inhibitor, cause a loss of H4K16ac. DNA damage induces the phosphorylation of Tip60 mediated by VRK1 in the chromatin fraction. VRK1 directly interacts with and phosphorylates Tip60. Furthermore, the phosphorylation of Tip60 induced by doxorubicin is lost by depletion of VRK1 in both ATM +/+ and ATM&minus, /&minus, cells. Kinase-active VRK1, but not kinase-dead VRK1, rescues Tip60 phosphorylation induced by DNA damage independently of ATM. The Tip60 phosphorylation by VRK1 is necessary for the activating acetylation of ATM, and subsequent ATM autophosphorylation, and both are lost by VRK1 depletion. These results support that the VRK1 chromatin kinase is an upstream regulator of the initial acetylation of histones, and an early step in DNA damage responses (DDR).

Resumen del trabajo presentado en 16th Aseica International Congress, celebrado en Valencia (España) del 06 al 08 de 2018., [Introduction]: Eukaryotic DNA is highly organized in a packaged nucleoprotein structure known as chromatin. The genomic information included in this chromatin must be maintained unalterable in order to confer a stable cell identity on tissues and prevent diseases development, including cancer. For that, cells have developed a complex DNA damage response (DDR) that ensures genome integrity in response to different types of DNA lesions. In this respect, epigenetics plays a crucial role at different levels of the DDR: first, the addition of different chemical modifications to specific histone residues enables chromatin decompaction and guarantees the access of DNArepair proteins to the lesion. Furthermore, other epigenetic modifications are essential for the proper progression of the DDR according to the type of DNA damage and the phase of the cell cycle in which that damage occurred. All these modifications (including acetylations, methylations, phosphorylations, ubiquitylations and others) are modulated by histone-modifying enzymes that belong to different families depending on the type of modification that they catalyze: histone acetyltransferases (HATs), deacetylases (HDACs), methyltransferases, demethylases, kinases, ubiquitin ligases, etc. However, how all these enzymes are coordinated in order to guarantee a proper DDR is not well-known., [Objective]: Our main aim is to study the mechanism responsible for regulating histone-modifying enzymes during this process. In this context, we hypothesize that the chromatin kinase VRK1 might be involved in the regulation of these histone-modifiers, based on current data that show that the depletion of this Ser-Thr kinase prevents the addition of different epigenetic modifications (such as the acetylation on the lysine 16 of histone H4) required for the initial decompaction of chromatin during the DDR. Given this, we have focused our objectives on analyzing specifically how VRK1 is regulating the activity of the histone acetyltransferase Tip60/KAT5, since it is the enzyme responsible for catalyzing this acetylation of H4K16., [Methods]: In order to study the role of VRK1 in the regulation of Tip60/KAT5 activity during DDR, cell cultures were treated with the chemotherapy drug doxorubicin 10 µM. After treatment, we use different experimental approaches, including Western-blotting and immunofluorescence assays, to analyze different Tip60 properties such as interaction with VRK1, phosphorylation state or stability. Furthermore, parallel experiments depleting VRK1 were also carried out to probe if this Tip60 properties change in absence of the kinase., [Results]: Throughout our study, we have observed that VRK1 and Tip60/KAT5 form a basal complex in which the kinase rapidly phosphorylates the acetyltransferase only when DNA damage is induced with doxorubicin. This phosphorylation seems to be important for Tip60 accumulation during the DDR, since VRK1 depletion reduces Tip60 stability and impairs its chromatin accumulation in response to DNA damage. Finally, we studied the mechanism by which VRK1-mediated phosphorylation of Tip60 prevents its degradation. In this context, we have shown that levels of ubiquitylated Tip60 are reduced during DDR, which supports the fact that Tip60 accumulates in response to DNA damage. However, this decrease of ubiquitylated Tip60 after doxorubicin treatment is not allowed when VRK1 is silenced, indicating that VRK1-mediated phosphorylation could block Tip60 ubiquitylation and subsequent degradation via proteasome pathway., [Conclusion]: Altogether, these results indicate that VRK1 may be a key component regulating Tip60/KAT5 activity, which could be essential for the development of a proper DNA damage response. From a clinical point of view, this approach can provide new insights on how to deal with chemotherapy resistance by developing VRK1 inhibitors that increase the DNA damage accumulation in cancer cells and improve the efficiency of chemotherapy drugs.

Resumen del póster presentado al 1st Joint Meeting of the French-Portuguese-Spanish Biochemical and Molecular Biology Societies y al XL Spanish Society of Biochemistry and Molecular Biology (SEBBM) Congress, celebrado en Barcelona (España) del 23 al 26 de octubre de 2017., Eukaryotic DNA is highly organized in a packaged nucleoprotein structure known as chromatin. This chromatin is subjected to dynamics that regulate its degree of compaction in processes such as cell proliferation or DNA damage repair. Chromatin dynamics are mainly governed by an epigenetic set of heritable chemical modifi cations that occurs in the tail of the histones. One of the most important epigenetic modifications is acetylation which, generally, allows chromatin decompacting of those regions that either need to be expressed during cell cycle or repaired. This modification is carried out by a family of enzymes called histone acetyl-transferases (HATs). However, the mechanism by which the activity of HATs is regulated in response to mitogens or DNA damage is unknown. In this context, a kinase could be responsible for activating these histone-modifiers, since it is well-known that phosphorylation is an important modification that regulates protein activity in many cellular processes. Among all the kinases present in cells, we hypothesize that the kinase VRK1 is a candidate to perform such a role, based on current data that supports that this Ser-Thr kinase has an important role in both DNA damage response and cell proliferation. To probe this hypothesis, we have studied the eff ect of VRK1 on Tip60, one of the most important HATs. Our results show that VRK1 interacts and phosphorylates Tip60 in response to both DNA damage and mitogens. Further, VRK1 knock-down induces Tip60 degradation, suggesting that this phosphorylation is important for Tip60 stability. Altogether these data indicates that VRK1 may be a key component in the regulation of Tip60 activity, which could play an important role in chromatin dynamics during both cell proliferation and DNA damage response.

Resumen del póster presentado a la EMBO Conference: "Chromatin and Epigenetics", celebrada en Heidelberg (Alemania) del 3 al 6 de mayo de 2017., Genome integrity is continuously challenged by endogenous and exogenous agents causing DNA damage. To counteract the adverse consequences associated to DNA lesions, eukaryotic cells have developed DNA damage signaling and repair machineries specifically adapted to the type of damage. Defects in DNA damage response contribute to aging and several disorders, including cancer and neurodegenerative diseases, which highlights their critical importance for cell viability. Double-strand breaks (DSBs), the most deleterious form of DNA damage, occur in the context of a highly organized chromatin environment. The dynamics of DNA repair proteins, which in many cases form detectable foci, are dictated by chromatin organization and transcriptional activity. Because of that, all eukaryotic DNA repair pathways need to unravel the compacted chromatin structure to facilitate access of the repair machinery and restoration of the original chromatin state afterwards. However, how accessibility and transcriptional silencing are orchestrated and interpreted at DSB sites is still unclear. In order to study the connection between chromatin structure alterations and DSB repair foci formation, pharmacological inhibitors of histone deacetylases, acetylases, methylases or demethylases were used in tumor cell lines before and after inducing DNA damage by both ionizing radiation or Olaparib treatment, widely employed in cancer therapies. Their effect on the assembly of DSB repair foci were assessed later, focusing on sensor and mediator proteins, such as γH2AX, Nbs1, MDC1 or 53BP1. Our results seem to indicate that some HAT and KMT inhibitors impair the formation of 53BP1 foci, not the γH2AX ones, at damage sites. This supports the idea that specific histone acetylations and methylations are necessary for the assembly or stability of 53BP1 foci in resting cells. Further experiments are being performed to determine the molecular mechanism and the modifications of histones implicated in this process.

Resumen del póster presentado a la EACR Conference: Protecting the Code, Epigenetic Impacts on Genome Stability, celebrado en berlin (Alemania) del 29 de octubre al 1 de noviembre de 2017., Double-strand breaks (DSBs) are the most deleterious form of DNA damage and they can arise from external and internal sources. In order to preserve their genome integrity, eukaryotic cells have developed DNA damage signaling and repair machineries whose deployment depends on the nature of the lesion. DNA damage takes place within the complex organization of chromatin, which acts as a barrier to the efficient detection and repair of DNA lesions. Because of that, all eukaryotic DSB repair pathways involve physiological chromatin alterations that facilitate the accessibility of repair machinery at damage sites and restore its local architecture afterwards. The current challenge is to understand how DNA repair occurs in the context of a highly organized chromatin environment. During DNA damage sensing and repair, histones undergo a set of posttranslational modifications (PTMs), including acetylation and methylation. Our aim is to determine how PTMs coordinate and amplify the DNA damage response (DDR). For this purpose, different tumor cell lines were incubated with pharmacological inhibitors of chromatin modifiers, followed by induction of DNA damage by either ionizing radiation or chemotherapeutic drugs like Olaparib. Next, DNA repair foci formation was assessed, focusing on sensor and mediator proteins, such as γ-H2AX, Nbs1, MDC1 or 53BP1. Interestingly, 53BP1 foci formation was impaired after inhibiting histone acetylases and/or methylases, while the assembly of γ-H2AX ones was not affected. This supports the idea that precise PMTs are absolutely essential to 53BP1 recruitment and, thus, to DNA repair. Based on these results, we hypothesize that specific inhibitors against epigenetic marks could have potential as anti-cancer therapies, since inaccurate and inefficient repair of DSBs and changes in acetylation/methylation states would contribute to cell death in tumors.

Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016., [Introduction]: Genome integrity is continuously challenged by exogenous and endogenous agents causing DNA damage, both in euchromatin and heterocromatin. The repair of these lesions involves changes in the histone organization, such as recruitment and/or incorporation of histone variants or covalent modifications of histones, in order to promote the formation of open, relaxed chromatin structues. Recently, a growing number of chromatin components, remodelers and modifications has been identificated as key players in DNA repair, emphasizing the complex role of chromatin in this process. On the other hand, defects in DNA repair pathways enable cancer cells to survive DNA damage. For this reason, the combination of DNA-damaging agents with specific inhibitors of these pathways could be used in cancer treatment. Currently, specific inhibitors against chromatin remodelers have been developed, but it is still unclear how they act in tumor cells. Our aim ls to detemine the effect of histone acetylation/deacetylation and methylation/demethylation inhibition on DNA repair foci. [Results and conclusion]: We use pharmacological inhibitors of HAIs (C646 and MGl49); HDACs (Entinostat and SAHA); KMTs (Chaetocin); and KDMs (JMJD2 inhibitor). Some of them are being employed in preclinical regulatory studies. In this analysis, we can observe that closed chromatin induced by HAIs inhibitors seems to affect the formation of yH2AX and 53BP1 foci, and the dynamics offoci formation corresponds with the increase of fluorescence level of H4 acetylation after inducing DNA damage. Based on these results, we conclude that chromatin relaxation is an essential early step in DNA repair, which can be blocked by specific inhibitors against HAIs, sensibilizing cells to treatments

Memoria presentada por el graduado en Biotecnología Raúl García González, que ha sido realizada en el Instituto de Biología Molecular y Celular del Cáncer para optar al título de Máster en Biología y Clínica del Cáncer por la Universidad de Salamanca., [ES]: El uso de la quimioterapia como tratamiento antitumoral ha mejorado la supervivencia de muchos pacientes con cáncer. Uno de los agentes quimioterapéuticos más utilizados a nivel clínico es la doxorrubicina. Este compuesto, perteneciente a la familia de las antraciclinas, inhibe la actividad de la topoisomerasa II, provocando con ello roturas de doble cadena en el ADN que pueden inducir la muerte celular. Desafortunadamente, la aplicación de este fármaco muchas veces cuenta con una serie de inconvenientes, como pueden ser la aparición de efectos secundarios de diversa consideración o el desarrollo de resistencia al mismo. Teniendo en cuenta esto, la búsqueda de nuevas estrategias terapéuticas capaces de disminuir la dosis de doxorrubicina y, al mismo tiempo, dificultar el desarrollo de resistencia por parte de las células tumorales constituye un reto para el ámbito científico. En relación a esto, en este trabajo se pretende estudiar la implicación que tiene la serina-treonina quinasa VRK1 en la reparación de roturas de doble cadena y, por consiguiente, en el desarrollo de resistencia por parte de las células tumorales., [EN]: Use of chemotherapy has improved survival for many patients with cancer. One of the most used chemotherapeutic agents is doxorubicin. This compound, belonging to the family of anthracyclines, is a topoisomerase-II inhibitor that causes double-strand breaks in the DNA and leads to cell death. Unfortunately, the application of this drug commonly shows some limitations, such as appearance of side effects or development of resistance. Given this, searching new therapeutic strategies aiming to reduce doxorubicin doses (and therefore side effects) and, at the same time, hindering the development of resistance has become a challenge for the scientific field. Based on this, this work intends to study the role of the serine-threonine kinase VRK1 in the response to double strand breaks and, thereby, in the development of resistance by tumor cells.

Gamma titanium aluminides are intermetallic alloys with great potential for aerospace applications in advanced aerospace engine and high-temperature airframe components and, in particular, in low pressure turbines (LPT), because they can provide increased thrust-to-weight ratios and improved efficiency under aggressive environments at temperatures up to 750 °C. Thus, - alloys are projected to replace the heavier -base superalloys currently used for LPT blades manufacturing. These alloys offer low density, high specific strength, creep and corrosion resistance at high temperatures. Figure 1 compares their mechanical behavior with that of other high temperature alloys at a wide range of temperatures. However, the wide commercialization of gamma titanium aluminides is prevented by their low ductility as well as by the lack of quantitative models relating the microstructure and the mechanical behavior. Moreover, their full potential has not been exploited because a thorough knowledge on the possibilities for microstructural development upon cooling from the liquid phase or from high temperature phase domains is still lacking. The aim of this project is to contribute to fill this knowledge gap by taking advantage of Gleeble technology and, in particular, of the possibility to use extremely high and well measured cooling rates to generate novel microstructures in a Ti-45Al-2Nb-Mn(at.%)+0.8(vol.%)TiB2 (Ti4522XD) alloy. The developed microstructures are thoroughly characterized by optical, scanning and transmission electron microscopy and a link with their mechanical behavior, evaluated by hardness measurements, is established. Ingeniería Aeroespacial