Strategies for modulating the process of partial reprogramming as an anti-aging tool
Authorship
R.P.P.
Master in Genomics and Genetics
R.P.P.
Master in Genomics and Genetics
Defense date
07.11.2024 16:00
07.11.2024 16:00
Summary
Aging is a degenerative process characterized by numerous hallmarks, among which the activation of the cellular senescence program stands out. This state is defined by a prolonged and stable cell cycle arrest in which cells remain metabolically active, releasing factors known as the Senescence-Associated Secretory Phenotype (SASP) into the environment. Cellular reprogramming, on the other hand, allows somatic cells to dedifferentiate into a pluripotent state through the expression of four transcription factors known as OSKM. Both processes are closely related, with senescence playing dual antagonistic roles. Senescence represents an intrinsic barrier against reprogramming, while the SASP enhances its efficiency. However, the effect that factors secreted by cells undergoing reprogramming have on senescent cells remains to be elucidated. Here we show that conditioned media derived from mouse embryonic fibroblasts undergoing reprogramming positively affects irradiation-induced senescent cells. This outcome is manifested as morphological changes, including a decrease in nucleus size, a reduction in Beta-galactosidase enzymatic activity associated with senescence, and an increase in cell viability. Additionally, we have studied whether the observed effect depends on the expression of the reprogramming factors or on the pluripotency state itself, showing OSKM as crucial to observe this effect. Therefore, these findings provide an indication of the potential of secreted factors from cells with OSKM expression as possible anti-aging tools.
Aging is a degenerative process characterized by numerous hallmarks, among which the activation of the cellular senescence program stands out. This state is defined by a prolonged and stable cell cycle arrest in which cells remain metabolically active, releasing factors known as the Senescence-Associated Secretory Phenotype (SASP) into the environment. Cellular reprogramming, on the other hand, allows somatic cells to dedifferentiate into a pluripotent state through the expression of four transcription factors known as OSKM. Both processes are closely related, with senescence playing dual antagonistic roles. Senescence represents an intrinsic barrier against reprogramming, while the SASP enhances its efficiency. However, the effect that factors secreted by cells undergoing reprogramming have on senescent cells remains to be elucidated. Here we show that conditioned media derived from mouse embryonic fibroblasts undergoing reprogramming positively affects irradiation-induced senescent cells. This outcome is manifested as morphological changes, including a decrease in nucleus size, a reduction in Beta-galactosidase enzymatic activity associated with senescence, and an increase in cell viability. Additionally, we have studied whether the observed effect depends on the expression of the reprogramming factors or on the pluripotency state itself, showing OSKM as crucial to observe this effect. Therefore, these findings provide an indication of the potential of secreted factors from cells with OSKM expression as possible anti-aging tools.
Direction
DA SILVA ALVAREZ, SABELA (Tutorships)
Collado Rodríguez, Manuel (Co-tutorships)
DA SILVA ALVAREZ, SABELA (Tutorships)
Collado Rodríguez, Manuel (Co-tutorships)
Court
RAMOS MARTINEZ, JUAN IGNACIO (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
Bouza Fernandez, M Carmen (Member)
RAMOS MARTINEZ, JUAN IGNACIO (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
Bouza Fernandez, M Carmen (Member)
Reclassification of Missense Variants of Uncertain Significance in the BRCA1 and BRCA2 Genes
Authorship
C.F.P.E.
Master in Genomics and Genetics
C.F.P.E.
Master in Genomics and Genetics
Defense date
07.11.2024 16:30
07.11.2024 16:30
Summary
The classification of genetic variants in hereditary cancer is a procedure routinely performed in genetic diagnostic laboratories. This classification is based on different types of evidence (e.g., type of variant, population frequency of the variant, pathogenicity prediction, functional impact, segregation of the variant in the disease, among others) that, when analyzed together, allow determining the impact of the variants on disease development. To determine the evidence to consider and the impact of each on the interpretation of genetic variants, both general guidelines and gene-specific guides have been developed. Recently, the ClinGen ENIGMA group (Evidence-based Network for the Interpretation of Germline Mutant Alleles) adapted the general standards and guidelines proposed in 2015 by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) to the BRCA1 and BRCA2 genes. The primary objective of this master final work was the reevaluation of 122 missense variants of uncertain significance identified in the BRCA1 and BRCA2 genes during genetic analysis at the Public Foundation of Genomic Medicine of Galicia (FPGMX), using the new ClinGen ENIGMA BRCA1 and BRCA2 ACMG/AMP guidelines. Additionally, we aimed to identify the most informative pieces of evidence for this reclassification. Following the application of the criteria proposed in these new guidelines, we reclassified 82% (100/122) of the initial variants of uncertain significance. Ninety-eight variants were classified as Benign and Likely Benign (15 and 83, respectively), and 2 variants were reclassified as Likely Pathogenic. Twenty-two variants remained as variants of uncertain significance. Among the most informative evidence for this reclassification were population frequency, variant position, and predicted impact.
The classification of genetic variants in hereditary cancer is a procedure routinely performed in genetic diagnostic laboratories. This classification is based on different types of evidence (e.g., type of variant, population frequency of the variant, pathogenicity prediction, functional impact, segregation of the variant in the disease, among others) that, when analyzed together, allow determining the impact of the variants on disease development. To determine the evidence to consider and the impact of each on the interpretation of genetic variants, both general guidelines and gene-specific guides have been developed. Recently, the ClinGen ENIGMA group (Evidence-based Network for the Interpretation of Germline Mutant Alleles) adapted the general standards and guidelines proposed in 2015 by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) to the BRCA1 and BRCA2 genes. The primary objective of this master final work was the reevaluation of 122 missense variants of uncertain significance identified in the BRCA1 and BRCA2 genes during genetic analysis at the Public Foundation of Genomic Medicine of Galicia (FPGMX), using the new ClinGen ENIGMA BRCA1 and BRCA2 ACMG/AMP guidelines. Additionally, we aimed to identify the most informative pieces of evidence for this reclassification. Following the application of the criteria proposed in these new guidelines, we reclassified 82% (100/122) of the initial variants of uncertain significance. Ninety-eight variants were classified as Benign and Likely Benign (15 and 83, respectively), and 2 variants were reclassified as Likely Pathogenic. Twenty-two variants remained as variants of uncertain significance. Among the most informative evidence for this reclassification were population frequency, variant position, and predicted impact.
Direction
CARRACEDO ALVAREZ, ANGEL MARIA (Tutorships)
Vega Gliemmo, Ana Paula (Co-tutorships)
CARRACEDO ALVAREZ, ANGEL MARIA (Tutorships)
Vega Gliemmo, Ana Paula (Co-tutorships)
Court
RAMOS MARTINEZ, JUAN IGNACIO (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
Bouza Fernandez, M Carmen (Member)
RAMOS MARTINEZ, JUAN IGNACIO (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
Bouza Fernandez, M Carmen (Member)
Evaluation of the regulation of the ribosomal protein RPL23 by ubiquitin-like proteins
Authorship
E.P.G.
Master in Genomics and Genetics
E.P.G.
Master in Genomics and Genetics
Defense date
07.11.2024 17:00
07.11.2024 17:00
Summary
Ribosomal proteins are proteins that, together with ribosomal RNA, constitute the ribosomes. In addition to their structural and functional role in protein synthesis, ribosomal proteins carry out multiple extra-ribosomal functions such as regulating cell growth and apoptosis. One of these proteins is the component of the large ribosomal subunit 60S RPL23. In addition to its ribosomal function, RPL23 was identified as a negative regulator of cellular apoptosis through the negative modulation of Miz-1-induced transcription of p21Cip1 and p15Ink4b, and as an activator of p53 in response to the oncogene RAS when the tumour suppressor ARF is not expressed. A significant increase in the levels of this protein in patients with different types of cancer is associated with the ability of tumour cells to evade apoptosis induced in response to various anti-tumor agents. The functional complexity of RPL23, capable of exerting both anti-tumour and pro-tumour functions, highlights the importance of a tight regulation of its activity. In this context, our hypothesis is that the RPL23 protein, similarly to RPL11 protein, could be regulated through its interaction with the ubiquitin-like proteins SUMO (small ubiquitin-like protein) and NEDD8 (neural precursor cell expressed, developmentally downregulated 8 ). Previous results from the laboratory suggest that RPL23 can be SUMOylated and NEDDylated in cells, but whether these modifications occur in vitro is still unclear. In addition, the molecular mechanisms that regulate these modifications and their impact on the protein's activities are unknown. This work aims to advance in the study of the regulation of RPL23 through its interaction with SUMO or NEDD8. Our results confirm that RPL23 is modified by SUMO and NEDD8. These modifications seem to respond to specific stimuli. Thus, overexpression of the RAS oncogene promotes NEDDylation of RPL23 and the formation of SUMO2 chains conjugated to the ribosomal protein. The modification of RPL23 by SUMO is also induced in response to treatment with the NEDDylation inhibitor MLN4924, suggesting that NEDD8 and SUMO might compete to bind to RPL23. In support of this hypothesis, we observed a reduction in the modification of RPL23 by SUMO2 or NEDD8 when both modifiers are present. To further investigate this hypothesis, we generated a mutant of RPL23 in one of the lysine residues that may function as SUMO and/or NEDD8 acceptor site and analyzed its SUMOylation and NEDDylation. The data obtained indicate that lysine K66 is a SUMOylation site, but its mutation does not inhibit the NEDDylation of the protein. Analysis of the impact of these modifications on the subcellular localization of the protein revealed that, unlike the wild-type RPL23 protein, which is mainly localized in the cell nucleolus, the RPL23-K66R SUMOylation mutant is localized in the nucleoplasm. A translocation of RPL23 from the nucleolus to the nucleoplasm was also observed when SUMO2 was overexpressed. These results led us to evaluate whether K66 was part of a potential nucleolar localization signal. In silico analysis suggests that, indeed, lysine K66 may be associated to a NLS, which could explain these results. SUMO conjugation to lysine K66 of RPL23 could prevent the functionality of the signal inhibiting RPL23 to enter into the nucleolus, similarly to the mutation of the lysine residue. Further studies are needed to confirm these results and to discover how these modifications influence the functionality of the protein. This knowledge could help control its activity and, therefore, would open a window of opportunity to act against those pathologies in which RPL23 is involved.
Ribosomal proteins are proteins that, together with ribosomal RNA, constitute the ribosomes. In addition to their structural and functional role in protein synthesis, ribosomal proteins carry out multiple extra-ribosomal functions such as regulating cell growth and apoptosis. One of these proteins is the component of the large ribosomal subunit 60S RPL23. In addition to its ribosomal function, RPL23 was identified as a negative regulator of cellular apoptosis through the negative modulation of Miz-1-induced transcription of p21Cip1 and p15Ink4b, and as an activator of p53 in response to the oncogene RAS when the tumour suppressor ARF is not expressed. A significant increase in the levels of this protein in patients with different types of cancer is associated with the ability of tumour cells to evade apoptosis induced in response to various anti-tumor agents. The functional complexity of RPL23, capable of exerting both anti-tumour and pro-tumour functions, highlights the importance of a tight regulation of its activity. In this context, our hypothesis is that the RPL23 protein, similarly to RPL11 protein, could be regulated through its interaction with the ubiquitin-like proteins SUMO (small ubiquitin-like protein) and NEDD8 (neural precursor cell expressed, developmentally downregulated 8 ). Previous results from the laboratory suggest that RPL23 can be SUMOylated and NEDDylated in cells, but whether these modifications occur in vitro is still unclear. In addition, the molecular mechanisms that regulate these modifications and their impact on the protein's activities are unknown. This work aims to advance in the study of the regulation of RPL23 through its interaction with SUMO or NEDD8. Our results confirm that RPL23 is modified by SUMO and NEDD8. These modifications seem to respond to specific stimuli. Thus, overexpression of the RAS oncogene promotes NEDDylation of RPL23 and the formation of SUMO2 chains conjugated to the ribosomal protein. The modification of RPL23 by SUMO is also induced in response to treatment with the NEDDylation inhibitor MLN4924, suggesting that NEDD8 and SUMO might compete to bind to RPL23. In support of this hypothesis, we observed a reduction in the modification of RPL23 by SUMO2 or NEDD8 when both modifiers are present. To further investigate this hypothesis, we generated a mutant of RPL23 in one of the lysine residues that may function as SUMO and/or NEDD8 acceptor site and analyzed its SUMOylation and NEDDylation. The data obtained indicate that lysine K66 is a SUMOylation site, but its mutation does not inhibit the NEDDylation of the protein. Analysis of the impact of these modifications on the subcellular localization of the protein revealed that, unlike the wild-type RPL23 protein, which is mainly localized in the cell nucleolus, the RPL23-K66R SUMOylation mutant is localized in the nucleoplasm. A translocation of RPL23 from the nucleolus to the nucleoplasm was also observed when SUMO2 was overexpressed. These results led us to evaluate whether K66 was part of a potential nucleolar localization signal. In silico analysis suggests that, indeed, lysine K66 may be associated to a NLS, which could explain these results. SUMO conjugation to lysine K66 of RPL23 could prevent the functionality of the signal inhibiting RPL23 to enter into the nucleolus, similarly to the mutation of the lysine residue. Further studies are needed to confirm these results and to discover how these modifications influence the functionality of the protein. This knowledge could help control its activity and, therefore, would open a window of opportunity to act against those pathologies in which RPL23 is involved.
Direction
Rivas Vázquez, María del Carmen (Tutorships)
ROBLEDO SANCHEZ, DIEGO (Co-tutorships)
Rivas Vázquez, María del Carmen (Tutorships)
ROBLEDO SANCHEZ, DIEGO (Co-tutorships)
Court
RAMOS MARTINEZ, JUAN IGNACIO (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
Bouza Fernandez, M Carmen (Member)
RAMOS MARTINEZ, JUAN IGNACIO (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
Bouza Fernandez, M Carmen (Member)
CRISPR-Cas9 gene editing of holm oak and chestnut somatic embryos
Authorship
P.M.P.I.
Master in Genomics and Genetics
P.M.P.I.
Master in Genomics and Genetics
Defense date
07.11.2024 17:30
07.11.2024 17:30
Summary
Holm oak (Quercus ilex) and chestnut (Castanea sativa) are two species of great social, economic and ecological importance in the Iberian Peninsula. In recent years, their populations have been decimated by the diseases holm oak decline and ink disease (chestnut), both caused by the infection of the soil-borne oomycete Phytophthora cinnamomi and accelerated by climate change. The measures taken so far have not been sufficient to control the advance of the disease, so generating plants with tolerance to the oomycete has become a priority. The long generation time and heterozygosity of these species severely limit the implementation of conventional breeding programmes, so the use of biotechnological tools may be a desirable alternative. Among these tools, gene editing with CRISPR/Cas9 stands out, which has revolutionised genetic engineering by allowing the introduction of point mutations and the introduction of new genetic variants through homology-directed recombination in a simple, flexible and efficient way. However, gene editing remains largely unexplored in forest species. In this work we have investigated gene editing in holm oak and chestnut, using somatic embryos as a way of regenerating the edited cells. In holm oak, as a proof of concept of gene editing, we sought to edit the gene encoding phytoene desaturase (PDS), whose mutation interrupts chlorophyll biosynthesis, giving rise to an albino phenotype, which allows visual assessment of the effectiveness of the editing. However, only albino callus was obtained, and we were unable to obtain somatic embryos with the edited PDS gene. In chestnut, we searched for and succeeded in editing the Downy Mildew Resistant 6 (DMR6) gene, a susceptibility gene (S gene) that pathogens use as a target to suppress plant immunity. Globular and early torpedo stage chestnut somatic embryos were co-cultured for 5 days with Agrobacterium carrying a CRISPR/Cas9 construct targeting DMR6 and subsequently grown on a selection medium containing kanamycin. After 10 weeks of subculture on selection medium, eleven kanamycin-resistant embryogenic lines were isolated. Genotyping of these lines through target Sanger sequencing revealed successful gene editing. Finally, by an in vitro infection assay with P. cinnamomi, the edited lines were found to show a higher tolerance to the oomycete than the unedited lines.
Holm oak (Quercus ilex) and chestnut (Castanea sativa) are two species of great social, economic and ecological importance in the Iberian Peninsula. In recent years, their populations have been decimated by the diseases holm oak decline and ink disease (chestnut), both caused by the infection of the soil-borne oomycete Phytophthora cinnamomi and accelerated by climate change. The measures taken so far have not been sufficient to control the advance of the disease, so generating plants with tolerance to the oomycete has become a priority. The long generation time and heterozygosity of these species severely limit the implementation of conventional breeding programmes, so the use of biotechnological tools may be a desirable alternative. Among these tools, gene editing with CRISPR/Cas9 stands out, which has revolutionised genetic engineering by allowing the introduction of point mutations and the introduction of new genetic variants through homology-directed recombination in a simple, flexible and efficient way. However, gene editing remains largely unexplored in forest species. In this work we have investigated gene editing in holm oak and chestnut, using somatic embryos as a way of regenerating the edited cells. In holm oak, as a proof of concept of gene editing, we sought to edit the gene encoding phytoene desaturase (PDS), whose mutation interrupts chlorophyll biosynthesis, giving rise to an albino phenotype, which allows visual assessment of the effectiveness of the editing. However, only albino callus was obtained, and we were unable to obtain somatic embryos with the edited PDS gene. In chestnut, we searched for and succeeded in editing the Downy Mildew Resistant 6 (DMR6) gene, a susceptibility gene (S gene) that pathogens use as a target to suppress plant immunity. Globular and early torpedo stage chestnut somatic embryos were co-cultured for 5 days with Agrobacterium carrying a CRISPR/Cas9 construct targeting DMR6 and subsequently grown on a selection medium containing kanamycin. After 10 weeks of subculture on selection medium, eleven kanamycin-resistant embryogenic lines were isolated. Genotyping of these lines through target Sanger sequencing revealed successful gene editing. Finally, by an in vitro infection assay with P. cinnamomi, the edited lines were found to show a higher tolerance to the oomycete than the unedited lines.
Direction
SAMPEDRO JIMÉNEZ, JAVIER (Tutorships)
Martínez Santiago, María Teresa (Co-tutorships)
Corredoira Castro, María Elena (Co-tutorships)
SAMPEDRO JIMÉNEZ, JAVIER (Tutorships)
Martínez Santiago, María Teresa (Co-tutorships)
Corredoira Castro, María Elena (Co-tutorships)
Court
RAMOS MARTINEZ, JUAN IGNACIO (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
Bouza Fernandez, M Carmen (Member)
RAMOS MARTINEZ, JUAN IGNACIO (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
Bouza Fernandez, M Carmen (Member)
Role of DYRK2 and PIM3 kinases in axonal regeneration after spinal cord injury in zebrafish
Authorship
A.B.S.L.
Master in Genomics and Genetics
A.B.S.L.
Master in Genomics and Genetics
Defense date
07.11.2024 18:00
07.11.2024 18:00
Summary
In humans, spinal cord injuries lead to a permanent disability. The lack of regeneration of descending axons after the injury is one of the main causes of the loss of function. In contrast to mammals, zebrafish adults and larvae can regenerate spinal cord descending axons after a complete spinal cord injury. A possible beneficial role of kinases DYRK2 and PIM3 in the promotion of axonal regeneration after spinal cord injury in fishes was detected in previous transcriptomic studies. Hence, the aim of this work is to study the role of kinases DYRK2 and PIM3 in axonal regeneration in zebrafish larvae after a complete spinal cord injury. For this study, pharmacological treatments with PIM3 kinase inhibitors and genetic treatments generating dyrk2 and pim3 Crispants were carried out. My results show that dyrk2 mutation in Crispant zebrafish embryos leads to a reduction in the thickness of the axonal bridge after a complete spinal cord transection. Similar results were obtained with pim3 Crispant embryos. Nonetheless, in both cases it would be appropriate to repeat the experiments with a higher sample size. Results obtained with pharmacological inhibitors also suggest that PIM3 specific inhibition leads to decreased regeneration of the axonal bridge. In conclusion, DYRK2 and PIM3 kinases seem to have an important role in promoting axon regeneration in zebrafish larvae. However, it would be of great interest to perform a detailed study of the signalling pathways and proteins phosphorylated by these kinases to better understand axonal regeneration mechanisms and, in the future, translate this information for the design of spinal cord therapies.
In humans, spinal cord injuries lead to a permanent disability. The lack of regeneration of descending axons after the injury is one of the main causes of the loss of function. In contrast to mammals, zebrafish adults and larvae can regenerate spinal cord descending axons after a complete spinal cord injury. A possible beneficial role of kinases DYRK2 and PIM3 in the promotion of axonal regeneration after spinal cord injury in fishes was detected in previous transcriptomic studies. Hence, the aim of this work is to study the role of kinases DYRK2 and PIM3 in axonal regeneration in zebrafish larvae after a complete spinal cord injury. For this study, pharmacological treatments with PIM3 kinase inhibitors and genetic treatments generating dyrk2 and pim3 Crispants were carried out. My results show that dyrk2 mutation in Crispant zebrafish embryos leads to a reduction in the thickness of the axonal bridge after a complete spinal cord transection. Similar results were obtained with pim3 Crispant embryos. Nonetheless, in both cases it would be appropriate to repeat the experiments with a higher sample size. Results obtained with pharmacological inhibitors also suggest that PIM3 specific inhibition leads to decreased regeneration of the axonal bridge. In conclusion, DYRK2 and PIM3 kinases seem to have an important role in promoting axon regeneration in zebrafish larvae. However, it would be of great interest to perform a detailed study of the signalling pathways and proteins phosphorylated by these kinases to better understand axonal regeneration mechanisms and, in the future, translate this information for the design of spinal cord therapies.
Direction
BARREIRO IGLESIAS, ANTON (Tutorships)
BARREIRO IGLESIAS, ANTON (Tutorships)
Court
RAMOS MARTINEZ, JUAN IGNACIO (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
Bouza Fernandez, M Carmen (Member)
RAMOS MARTINEZ, JUAN IGNACIO (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
Bouza Fernandez, M Carmen (Member)
Xenotransplant assays in zebrafish as a model for the study of primary glioblastoma tumours.
Authorship
M.B.T.C.
Master in Genomics and Genetics
M.B.T.C.
Master in Genomics and Genetics
Defense date
07.11.2024 18:30
07.11.2024 18:30
Summary
Glioblastoma multiforme (GBM) is one of the most common malignant brain tumours. It is characterised as a stage IV primary tumour of astrocytic origin, for which most available treatments have limited efficacy. As a result, with an average patient survival of approximately 14.6 months, this type of cancer has a very poor prognosis. The project is focused on modelling primary GBM tumours in zebrafish (Danio rerio) with the aim of evaluating the efficacy of conventional drugs and thus determining if this model can be established as a useful tool in the study of this type of carcinoma. The project will begin with an initial assessment of the toxicity of conventional chemotherapeutics, such as temozolomide (TMZ), on zebrafish embryos. Subsequently, xenotransplants will be carried out using glioblastoma cells previously cultured in vitro. This process will allow the monitoring of tumour progression within zebrafish embryos once they have been exposed to the drug. It is hoped that the results of this project will provide valuable information on the utility of the zebrafish model in the study of GBM and, at the same time, will enable the design of possible therapeutic strategies that improve the prognosis of patients affected by this deadly disease.
Glioblastoma multiforme (GBM) is one of the most common malignant brain tumours. It is characterised as a stage IV primary tumour of astrocytic origin, for which most available treatments have limited efficacy. As a result, with an average patient survival of approximately 14.6 months, this type of cancer has a very poor prognosis. The project is focused on modelling primary GBM tumours in zebrafish (Danio rerio) with the aim of evaluating the efficacy of conventional drugs and thus determining if this model can be established as a useful tool in the study of this type of carcinoma. The project will begin with an initial assessment of the toxicity of conventional chemotherapeutics, such as temozolomide (TMZ), on zebrafish embryos. Subsequently, xenotransplants will be carried out using glioblastoma cells previously cultured in vitro. This process will allow the monitoring of tumour progression within zebrafish embryos once they have been exposed to the drug. It is hoped that the results of this project will provide valuable information on the utility of the zebrafish model in the study of GBM and, at the same time, will enable the design of possible therapeutic strategies that improve the prognosis of patients affected by this deadly disease.
Direction
CABEZAS SAINZ, PABLO (Tutorships)
CABEZAS SAINZ, PABLO (Tutorships)
Court
RAMOS MARTINEZ, JUAN IGNACIO (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
Bouza Fernandez, M Carmen (Member)
RAMOS MARTINEZ, JUAN IGNACIO (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
Bouza Fernandez, M Carmen (Member)
Search for genetic variants of familial heart diseases associated with the risk of ventricular dysfunction secondary to antitumor treatments.
Authorship
P.B.M.
Master in Genomics and Genetics
P.B.M.
Master in Genomics and Genetics
Defense date
07.11.2024 10:00
07.11.2024 10:00
Summary
Cardiovascular toxicity related to cancer treatment, or cardiotoxicity, is the name given to the set of cardiovascular manifestations derived from antitumor treatments. These adverse effects are increasingly relevant due to the growing survival of cancer patients and the expectations of an increase in cancer cases in the future. Therefore, it is of great importance to know the factors that contribute to the development of cardiotoxicity to improve the risk stratification of cancer patients. Currently, the variety of risk factors considered by risk stratification tools does not include genetic variability. However, several studies have described loci and genetic variants that could be related to the development of cardiotoxicity. This study aims to search for genetic variants of heart disease that may be associated with the risk of cardiovascular toxicity related to cancer treatment. To achieve this, an exome analysis was carried out on 127 oncological patients from the CARDIOTOX registry, encompassing individuals with and without cardiotoxicity, and the genetic variants in 536 genes associated with heart disease were prioritized and classified according to their pathogenicity. 11 pathogenic or probably pathogenic genetic variants associated with cardiac pathologies were found, in six main genes (TTN, ALPK3, KNCH2, MYOT, RIT1 and RBM20) and one secondary gene (NRAS) in their association with said pathologies in the study cohort. These genetic variants were observed in 5 individuals without cardiotoxicity and 4 individuals with cardiotoxicity, representing 4.40% and 13.89% of the patients in each group, respectively. Therefore, a higher prevalence of genetic variants in genes associated with heart disease was not observed among individuals who developed cardiotoxicity, nor in the most severe degrees of this condition. However, the high frequency of truncating variants in the TTN gene in these patients stands out, so these could play an important role in the predisposition to cardiotoxicity. Furthermore, individuals with cardiotoxicity who presented genetic variants associated with cardiac pathologies were entirely men, despite there being a greater proportion of women in the study cohort; this suggests that there may also be a sex difference in genetic susceptibility to cardiotoxicity.
Cardiovascular toxicity related to cancer treatment, or cardiotoxicity, is the name given to the set of cardiovascular manifestations derived from antitumor treatments. These adverse effects are increasingly relevant due to the growing survival of cancer patients and the expectations of an increase in cancer cases in the future. Therefore, it is of great importance to know the factors that contribute to the development of cardiotoxicity to improve the risk stratification of cancer patients. Currently, the variety of risk factors considered by risk stratification tools does not include genetic variability. However, several studies have described loci and genetic variants that could be related to the development of cardiotoxicity. This study aims to search for genetic variants of heart disease that may be associated with the risk of cardiovascular toxicity related to cancer treatment. To achieve this, an exome analysis was carried out on 127 oncological patients from the CARDIOTOX registry, encompassing individuals with and without cardiotoxicity, and the genetic variants in 536 genes associated with heart disease were prioritized and classified according to their pathogenicity. 11 pathogenic or probably pathogenic genetic variants associated with cardiac pathologies were found, in six main genes (TTN, ALPK3, KNCH2, MYOT, RIT1 and RBM20) and one secondary gene (NRAS) in their association with said pathologies in the study cohort. These genetic variants were observed in 5 individuals without cardiotoxicity and 4 individuals with cardiotoxicity, representing 4.40% and 13.89% of the patients in each group, respectively. Therefore, a higher prevalence of genetic variants in genes associated with heart disease was not observed among individuals who developed cardiotoxicity, nor in the most severe degrees of this condition. However, the high frequency of truncating variants in the TTN gene in these patients stands out, so these could play an important role in the predisposition to cardiotoxicity. Furthermore, individuals with cardiotoxicity who presented genetic variants associated with cardiac pathologies were entirely men, despite there being a greater proportion of women in the study cohort; this suggests that there may also be a sex difference in genetic susceptibility to cardiotoxicity.
Direction
CARRACEDO ALVAREZ, ANGEL MARIA (Tutorships)
Blanco Verea, Alejandro José (Co-tutorships)
BRION MARTINEZ, M. JOSE (Co-tutorships)
CARRACEDO ALVAREZ, ANGEL MARIA (Tutorships)
Blanco Verea, Alejandro José (Co-tutorships)
BRION MARTINEZ, M. JOSE (Co-tutorships)
Court
Bouza Fernandez, M Carmen (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
GÓMEZ PARDO, MARÍA BELÉN (Member)
Bouza Fernandez, M Carmen (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
GÓMEZ PARDO, MARÍA BELÉN (Member)
Deciphering Lynch-like syndrome
Authorship
O.G.E.
Master in Genomics and Genetics
O.G.E.
Master in Genomics and Genetics
Defense date
07.11.2024 10:30
07.11.2024 10:30
Summary
Lynch syndrome is a genetic disorder with autosomal dominant inheritance caused by pathogenic germline variants in the DNA mismatch repair (MMR) genes: MLH1, MSH2, MSH6, and PMS2. This syndrome predisposes individuals to an increased risk of developing colon and endometrial cancer, among others, throughout their lives. Lynch tumours are characterised by immunohistochemical expression deficiency in some of the MMR proteins and microsatellite instability (MSI). The use of genetic variant classification guidelines that are specific to MMR genes is essential for the diagnosis of Lynch syndrome. Lynch-like syndrome refers to those individuals who display deficient MMR tumours without MLH1 promoter methylation, but in whom no pathogenic germline variants in the MMR genes are identified. Lynch-like syndrome comprises a heterogeneous group, as the cause of loss of expression in MMR proteins can be either germline, and therefore hereditary, or somatic. The main aim of this study is the molecular characterisation of 122 individuals with suspected Lynch syndrome, 76% of whom were categorised as Lynch-like syndrome. By MS-MLPA, a somatic MLH1 hypermethylation study was conducted, which allowed 16 Lynch-like individuals to be categorised as sporadic cases. In addition, using classification guidelines developed by the ClinGen/InSiGHT ACMG MMR v1 expert panel, 62,5% of variants of uncertain significance (VUS) were reclassified as either pathogenic or probably pathogenic in 7 individuals from 3 unrelated families, or probably benign in another individual from the cohort. In this way, we were able to molecularly characterise part of the study cohort into cases with Lynch syndrome (29,5%) and cases with a sporadic origin (13,1%). However, 57,4% of individuals remained categorised as Lynch-like syndrome, for whom it would be advisable to perform somatic MMR sequencing to search biallelic mutations in the MMR genes, allowing a better characterisation of this syndrome.
Lynch syndrome is a genetic disorder with autosomal dominant inheritance caused by pathogenic germline variants in the DNA mismatch repair (MMR) genes: MLH1, MSH2, MSH6, and PMS2. This syndrome predisposes individuals to an increased risk of developing colon and endometrial cancer, among others, throughout their lives. Lynch tumours are characterised by immunohistochemical expression deficiency in some of the MMR proteins and microsatellite instability (MSI). The use of genetic variant classification guidelines that are specific to MMR genes is essential for the diagnosis of Lynch syndrome. Lynch-like syndrome refers to those individuals who display deficient MMR tumours without MLH1 promoter methylation, but in whom no pathogenic germline variants in the MMR genes are identified. Lynch-like syndrome comprises a heterogeneous group, as the cause of loss of expression in MMR proteins can be either germline, and therefore hereditary, or somatic. The main aim of this study is the molecular characterisation of 122 individuals with suspected Lynch syndrome, 76% of whom were categorised as Lynch-like syndrome. By MS-MLPA, a somatic MLH1 hypermethylation study was conducted, which allowed 16 Lynch-like individuals to be categorised as sporadic cases. In addition, using classification guidelines developed by the ClinGen/InSiGHT ACMG MMR v1 expert panel, 62,5% of variants of uncertain significance (VUS) were reclassified as either pathogenic or probably pathogenic in 7 individuals from 3 unrelated families, or probably benign in another individual from the cohort. In this way, we were able to molecularly characterise part of the study cohort into cases with Lynch syndrome (29,5%) and cases with a sporadic origin (13,1%). However, 57,4% of individuals remained categorised as Lynch-like syndrome, for whom it would be advisable to perform somatic MMR sequencing to search biallelic mutations in the MMR genes, allowing a better characterisation of this syndrome.
Direction
CARRACEDO ALVAREZ, ANGEL MARIA (Tutorships)
Ruiz Ponte, Clara María (Co-tutorships)
CARRACEDO ALVAREZ, ANGEL MARIA (Tutorships)
Ruiz Ponte, Clara María (Co-tutorships)
Court
Bouza Fernandez, M Carmen (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
GÓMEZ PARDO, MARÍA BELÉN (Member)
Bouza Fernandez, M Carmen (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
GÓMEZ PARDO, MARÍA BELÉN (Member)
Effect of listening to music on the salivary transcriptome of age-related cognitive disorders patients and healthy controls
Authorship
N.E.Z.M.
Master in Genomics and Genetics
N.E.Z.M.
Master in Genomics and Genetics
Defense date
07.11.2024 11:00
07.11.2024 11:00
Summary
Background and objective: Using music interventions as a non-pharmacological therapy to ameliorate cognitive and behavioral symptoms in age-related cognitive disorder (ACD) patients has gained popularity in recent years, but the evidence for their effectiveness remains inconsistent. The main objective is to explore the influence of musical stimuli on the salivary transcriptome of patients of ACD. Methods: Saliva samples were collected from 24 participants (14 healthy controls HC and 10 diagnosed with ACD) before and after the musical stimuli (n= 48 samples). RNA from saliva was isolated and analyzed using the nCounter NanoString technology. For the statistical analysis, the statistical computing platform R version 4.3.3. was employed. A paired-sampling design was used to carry out the transcriptome differences analysis before (timepoint 1, TP1) and after (timepoint 2, TP2) the musical stimuli. Results: The number of differentially expressed genes (DEGs) of ACD patients (n=31) was higher than the DEGs of the control group (n=7), which indicates that the upregulation is prominent in ACD patients. The DEGs transcriptome profile revealed a straightforward recognizable differentiation between TP1 and TP2 in ACD group, which can be immediately detected from the principal components PC1 (accounting for 79% variation) and PC2 (accounting for 13% variation). In ACD patients, CXCL8 was the top upregulated gene while LGALS3 was the top downregulated gene. Regarding the control group, THOP1 was the top downregulated gene. These genes play important roles in normal brain functions and are also altered in neurodegenerative conditions. Weighted Gene Co-expression Network Analysis reveals relevant and significant modules, both positive and negative correlated with music, implicated in neurodegenerative and immunological processes. Conclusion: This study presents a pioneering evidence of the impact of brief exposure to music on the salivary transcriptome in both ACD and HC participants. It sheds light on the potential of musical stimuli in affecting the gene expression, focusing on the entangled and complicated relationship between music and molecular responses in the human body.
Background and objective: Using music interventions as a non-pharmacological therapy to ameliorate cognitive and behavioral symptoms in age-related cognitive disorder (ACD) patients has gained popularity in recent years, but the evidence for their effectiveness remains inconsistent. The main objective is to explore the influence of musical stimuli on the salivary transcriptome of patients of ACD. Methods: Saliva samples were collected from 24 participants (14 healthy controls HC and 10 diagnosed with ACD) before and after the musical stimuli (n= 48 samples). RNA from saliva was isolated and analyzed using the nCounter NanoString technology. For the statistical analysis, the statistical computing platform R version 4.3.3. was employed. A paired-sampling design was used to carry out the transcriptome differences analysis before (timepoint 1, TP1) and after (timepoint 2, TP2) the musical stimuli. Results: The number of differentially expressed genes (DEGs) of ACD patients (n=31) was higher than the DEGs of the control group (n=7), which indicates that the upregulation is prominent in ACD patients. The DEGs transcriptome profile revealed a straightforward recognizable differentiation between TP1 and TP2 in ACD group, which can be immediately detected from the principal components PC1 (accounting for 79% variation) and PC2 (accounting for 13% variation). In ACD patients, CXCL8 was the top upregulated gene while LGALS3 was the top downregulated gene. Regarding the control group, THOP1 was the top downregulated gene. These genes play important roles in normal brain functions and are also altered in neurodegenerative conditions. Weighted Gene Co-expression Network Analysis reveals relevant and significant modules, both positive and negative correlated with music, implicated in neurodegenerative and immunological processes. Conclusion: This study presents a pioneering evidence of the impact of brief exposure to music on the salivary transcriptome in both ACD and HC participants. It sheds light on the potential of musical stimuli in affecting the gene expression, focusing on the entangled and complicated relationship between music and molecular responses in the human body.
Direction
SALAS ELLACURIAGA, ANTONIO (Tutorships)
SALAS ELLACURIAGA, ANTONIO (Tutorships)
Court
Bouza Fernandez, M Carmen (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
GÓMEZ PARDO, MARÍA BELÉN (Member)
Bouza Fernandez, M Carmen (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
GÓMEZ PARDO, MARÍA BELÉN (Member)
Molecular basis of lipomatosis associated with Familial Partial Lipodystrophy
Authorship
L.R.S.
Master in Genomics and Genetics
L.R.S.
Master in Genomics and Genetics
Defense date
07.11.2024 11:30
07.11.2024 11:30
Summary
Some Mendelian lipodystrophies are associated with the presence of lipomas, even in lipoatrophic regions. These are linked to variants in several genes, including the LMNA gene. Currently, the mechanisms involved in the abnormal proliferation of adipocytes in these neoplastic lesions are unknown. In the case of laminopathic lipodystrophies (familial partial lipodystrophy type 2 or FPLD2), the defect in the nuclear lamin A leads to an alteration in adipogenesis depending on the anatomical region (lipoatrophy in the extremities and buttocks, and lipohypertrophy in the face, neck, armpits, and interscapular region). There is currently evidence that in lipohypertrophic regions there is a process of transdifferentiation from brown to white fat, possibly mediated by the aldosterone receptor, to which progesterone also binds. Additionally, the hypothesis is that the development of lipomatous masses in patients with pathogenic LMNA variants could be mediated by alterations in proteins that regulate the cell cycle (p53, p21, Rb). Thus, three cell lines of preadipocytes (the fat of a control patient, the fat and the lipoma of a FPLD2 patient) are cultured with six treatments testing pre- and postmenopausal doses of beta-estradiol and progesterone. Proteins are then extracted from each line and treatment, and the levels of each protein are measured by Western blot.
Some Mendelian lipodystrophies are associated with the presence of lipomas, even in lipoatrophic regions. These are linked to variants in several genes, including the LMNA gene. Currently, the mechanisms involved in the abnormal proliferation of adipocytes in these neoplastic lesions are unknown. In the case of laminopathic lipodystrophies (familial partial lipodystrophy type 2 or FPLD2), the defect in the nuclear lamin A leads to an alteration in adipogenesis depending on the anatomical region (lipoatrophy in the extremities and buttocks, and lipohypertrophy in the face, neck, armpits, and interscapular region). There is currently evidence that in lipohypertrophic regions there is a process of transdifferentiation from brown to white fat, possibly mediated by the aldosterone receptor, to which progesterone also binds. Additionally, the hypothesis is that the development of lipomatous masses in patients with pathogenic LMNA variants could be mediated by alterations in proteins that regulate the cell cycle (p53, p21, Rb). Thus, three cell lines of preadipocytes (the fat of a control patient, the fat and the lipoma of a FPLD2 patient) are cultured with six treatments testing pre- and postmenopausal doses of beta-estradiol and progesterone. Proteins are then extracted from each line and treatment, and the levels of each protein are measured by Western blot.
Direction
SANCHEZ IGLESIAS, SOFIA (Tutorships)
ARAUJO VILAR, DAVID (Co-tutorships)
SANCHEZ IGLESIAS, SOFIA (Tutorships)
ARAUJO VILAR, DAVID (Co-tutorships)
Court
Bouza Fernandez, M Carmen (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
GÓMEZ PARDO, MARÍA BELÉN (Member)
Bouza Fernandez, M Carmen (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
GÓMEZ PARDO, MARÍA BELÉN (Member)
Characterization of the effect of new epigenetic therapies in cancer treatment through in vitro assays.
Authorship
X.R.A.V.
Master in Genomics and Genetics
X.R.A.V.
Master in Genomics and Genetics
Defense date
07.11.2024 13:00
07.11.2024 13:00
Summary
Renal cancer ranks ninth among the most common cancers in Spain, with a five-year survival rate of 17% in advanced stages. Immunotherapy has recently emerged as a promising option, with some patients achieving sustained responses over time; however, the percentage remains low. Therefore, there is an urgent need to find new therapies to increase its efficiency. In this context, we explored the potential of epigenetic therapies through in vitro assays, using epidrugs such as vorinostat (a histone deacetylase inhibitor) and azacitidine (a DNA methyltransferase inhibitor) in renal cancer cell lines (786-O and Caki-2). Our results revealed that these treatments induce both phenotypic and gene expression changes in tumor cells, affecting the cell cycle, promoting mesenchymal characteristics, and altering the expression of transposable elements (TEs) and the histone-lysine methyltransferase gene (EZH2). These findings support the potential of these new epigenetic therapies and the need for further research to increase the number of patients who can benefit from immunotherapy.
Renal cancer ranks ninth among the most common cancers in Spain, with a five-year survival rate of 17% in advanced stages. Immunotherapy has recently emerged as a promising option, with some patients achieving sustained responses over time; however, the percentage remains low. Therefore, there is an urgent need to find new therapies to increase its efficiency. In this context, we explored the potential of epigenetic therapies through in vitro assays, using epidrugs such as vorinostat (a histone deacetylase inhibitor) and azacitidine (a DNA methyltransferase inhibitor) in renal cancer cell lines (786-O and Caki-2). Our results revealed that these treatments induce both phenotypic and gene expression changes in tumor cells, affecting the cell cycle, promoting mesenchymal characteristics, and altering the expression of transposable elements (TEs) and the histone-lysine methyltransferase gene (EZH2). These findings support the potential of these new epigenetic therapies and the need for further research to increase the number of patients who can benefit from immunotherapy.
Direction
VERA RODRIGUEZ, MANUEL (Tutorships)
MARTINEZ FERNANDEZ, MONICA (Co-tutorships)
VERA RODRIGUEZ, MANUEL (Tutorships)
MARTINEZ FERNANDEZ, MONICA (Co-tutorships)
Court
Bouza Fernandez, M Carmen (Chairman)
MARTINEZ PORTELA, PAULINO (Secretary)
GÓMEZ PARDO, MARÍA BELÉN (Member)
Bouza Fernandez, M Carmen (Chairman)
MARTINEZ PORTELA, PAULINO (Secretary)
GÓMEZ PARDO, MARÍA BELÉN (Member)
Genomic analyses in natural populations of Arnica montana L.
Authorship
F.C.P.
Master in Genomics and Genetics
F.C.P.
Master in Genomics and Genetics
Defense date
07.11.2024 12:30
07.11.2024 12:30
Summary
Arnica montana L. is a perennial plant belonging to the Asteraceae family distributed along Europe. The species has pharmaceutical interest, due to its anti-inflammatory properties provided by some secondary metabolites, basically sesquiterpene lactones. The unregulated recollection in rural areas for these properties, alongside changes to their habitats, have decreased their populations, dramatically in some areas of Central Europe. Despite being classified in the IUCN Red List as Least Concern, the species is currently monitored and included in local red lists from several countries, being considered a species of interest by the European Union. Previous studies have focused on determining its population structure and the differences between two subspecies: A. montana subsp. montana and A. montana subsp. atlantica, with the latter appearing exclusively on lower altitude locations along the northern part of the Iberian Peninsula and southern France. These two subspecies have been linked to two different chemotypes according to the content of sesquiterpene lactones, with A. montana subsp. montana having more anti-inflammatory power but also being linked to higher allergenic responses. The aim of this study was to perform the first genomic approach in A. montana to: (i) conduct an analysis of population genomics and detect possible natural selection footprints; (ii) compare and discuss the results with previously published studies; (iii) detect SNPs with highly differentiated allelic frequencies between the two previously described chemotypes; and (iv) design of management and conservation strategies for A. montana in its natural habitats. For the study, samples were taken from 12 locations in northern Spain and genotyped using 2b-RAD, obtaining a panel of 5,675 SNPs. These locations showed a low genetic diversity and effective size, together with high differentiation among them, consistent with the results from previous studies. Potential outliers under selection were found, which might be involved in local adaptation, as well as outliers potentially involved in chemotype differentiation. Nevertheless, these findings ought to be validated in further studies using the A. montana reference genome, new biochemical data and common garden experiments. The creation of stocks for each of the defined management units would be recommended for the conservation of A. montana natural populations, for both restocking in existing locations as well as reintroduction in historical ones. Nonetheless, maintenance of the ecological quality in its wild habitats is also necessary for the viability of these populations, as well as promoting its crop to avoid overexploitation.
Arnica montana L. is a perennial plant belonging to the Asteraceae family distributed along Europe. The species has pharmaceutical interest, due to its anti-inflammatory properties provided by some secondary metabolites, basically sesquiterpene lactones. The unregulated recollection in rural areas for these properties, alongside changes to their habitats, have decreased their populations, dramatically in some areas of Central Europe. Despite being classified in the IUCN Red List as Least Concern, the species is currently monitored and included in local red lists from several countries, being considered a species of interest by the European Union. Previous studies have focused on determining its population structure and the differences between two subspecies: A. montana subsp. montana and A. montana subsp. atlantica, with the latter appearing exclusively on lower altitude locations along the northern part of the Iberian Peninsula and southern France. These two subspecies have been linked to two different chemotypes according to the content of sesquiterpene lactones, with A. montana subsp. montana having more anti-inflammatory power but also being linked to higher allergenic responses. The aim of this study was to perform the first genomic approach in A. montana to: (i) conduct an analysis of population genomics and detect possible natural selection footprints; (ii) compare and discuss the results with previously published studies; (iii) detect SNPs with highly differentiated allelic frequencies between the two previously described chemotypes; and (iv) design of management and conservation strategies for A. montana in its natural habitats. For the study, samples were taken from 12 locations in northern Spain and genotyped using 2b-RAD, obtaining a panel of 5,675 SNPs. These locations showed a low genetic diversity and effective size, together with high differentiation among them, consistent with the results from previous studies. Potential outliers under selection were found, which might be involved in local adaptation, as well as outliers potentially involved in chemotype differentiation. Nevertheless, these findings ought to be validated in further studies using the A. montana reference genome, new biochemical data and common garden experiments. The creation of stocks for each of the defined management units would be recommended for the conservation of A. montana natural populations, for both restocking in existing locations as well as reintroduction in historical ones. Nonetheless, maintenance of the ecological quality in its wild habitats is also necessary for the viability of these populations, as well as promoting its crop to avoid overexploitation.
Direction
VERA RODRIGUEZ, MANUEL (Tutorships)
CASANOVA CHICLANA, ADRIAN (Co-tutorships)
VERA RODRIGUEZ, MANUEL (Tutorships)
CASANOVA CHICLANA, ADRIAN (Co-tutorships)
Court
Bouza Fernandez, M Carmen (Chairman)
MARTINEZ PORTELA, PAULINO (Secretary)
GÓMEZ PARDO, MARÍA BELÉN (Member)
Bouza Fernandez, M Carmen (Chairman)
MARTINEZ PORTELA, PAULINO (Secretary)
GÓMEZ PARDO, MARÍA BELÉN (Member)
Analysis of epigenetic biomarkers in circulating DNA with clinical utility in colorectal cancer
Authorship
J.D.V.
Master in Genomics and Genetics
J.D.V.
Master in Genomics and Genetics
Defense date
07.11.2024 12:00
07.11.2024 12:00
Summary
Colorectal cancer (CRC) is the third most diagnosed cancer worldwide with 1.93 million new cases in 2020 and the second deadliest with nearly 1 million new deaths. Given its high prevalence and mortality rate, it is a priority to search for biomarkers for early diagnosis and prognosis that enable earlier treatment and better patient management, thereby reducing the number of deaths. This study focuses on identifying epigenetic markers, specifically DNA methylation, to improve non-invasive detection and management of CRC patients. To achieve this goal, circulating free DNA (cfDNA) present in liquid biopsies was characterized using epigenomic sequencing and digital PCR techniques. As a result, a non-invasive epigenetic signature capable of detecting CRC was identified. Among the genes in this signature, the methylation status of USP44 and SPOCK1 was validated, which were found to be hypermethylated in CRC cfDNA compared to healthy controls. The hypermethylation of these genes was also present in CRC tissue samples, where it showed an inverse relationship with their expression status. The methylation of USP44 and SPOCK1 in cfDNA proved useful for CRC detection and as a prognostic factor for survival. Overall, this study has identified and validated new non-invasive epigenetic biomarkers in liquid biopsies that hold great potential for the detection and prognosis of CRC.
Colorectal cancer (CRC) is the third most diagnosed cancer worldwide with 1.93 million new cases in 2020 and the second deadliest with nearly 1 million new deaths. Given its high prevalence and mortality rate, it is a priority to search for biomarkers for early diagnosis and prognosis that enable earlier treatment and better patient management, thereby reducing the number of deaths. This study focuses on identifying epigenetic markers, specifically DNA methylation, to improve non-invasive detection and management of CRC patients. To achieve this goal, circulating free DNA (cfDNA) present in liquid biopsies was characterized using epigenomic sequencing and digital PCR techniques. As a result, a non-invasive epigenetic signature capable of detecting CRC was identified. Among the genes in this signature, the methylation status of USP44 and SPOCK1 was validated, which were found to be hypermethylated in CRC cfDNA compared to healthy controls. The hypermethylation of these genes was also present in CRC tissue samples, where it showed an inverse relationship with their expression status. The methylation of USP44 and SPOCK1 in cfDNA proved useful for CRC detection and as a prognostic factor for survival. Overall, this study has identified and validated new non-invasive epigenetic biomarkers in liquid biopsies that hold great potential for the detection and prognosis of CRC.
Direction
Bouza Fernandez, M Carmen (Tutorships)
Díaz Lagares, Ángel (Co-tutorships)
Bouza Fernandez, M Carmen (Tutorships)
Díaz Lagares, Ángel (Co-tutorships)
Court
MARTINEZ PORTELA, PAULINO (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
GÓMEZ PARDO, MARÍA BELÉN (Member)
MARTINEZ PORTELA, PAULINO (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
GÓMEZ PARDO, MARÍA BELÉN (Member)
Detection of Dispaired Rhythmic Genes in a Mouse Model Lacking Astrocytic BMAL1 and its Relation with Circadiam Timekeeper System.
Authorship
E.E.E.B.
Master in Genomics and Genetics
E.E.E.B.
Master in Genomics and Genetics
Defense date
09.11.2024 10:00
09.11.2024 10:00
Summary
The purpose for this Master's thesis dissertation is to analyze the RNAseq and miRNome dataset obtained from mice lacking the gene BMAL1 in astrocytes (BMAL1cKO) and control animals at different moments of the day. The main objective is to identify differential expressed genes (DEGs) and miRNAs altered and their possible gene targets using RNAseq data. Furthermore, the results will provide key information about the way BMAL1 absence affects the cicadian timekeeping system at a molecular level, contributing to the knowledge of circadian rhythms genetics and the role of astrocytes in this system.
The purpose for this Master's thesis dissertation is to analyze the RNAseq and miRNome dataset obtained from mice lacking the gene BMAL1 in astrocytes (BMAL1cKO) and control animals at different moments of the day. The main objective is to identify differential expressed genes (DEGs) and miRNAs altered and their possible gene targets using RNAseq data. Furthermore, the results will provide key information about the way BMAL1 absence affects the cicadian timekeeping system at a molecular level, contributing to the knowledge of circadian rhythms genetics and the role of astrocytes in this system.
Direction
BARCA MAYO, OLGA (Tutorships)
BARCA MAYO, OLGA (Tutorships)
Court
Bouza Fernandez, M Carmen (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
DÍAZ FERNÁNDEZ, PABLO (Member)
Bouza Fernandez, M Carmen (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
DÍAZ FERNÁNDEZ, PABLO (Member)
Final Project
Authorship
R.P.V.
Master in Genomics and Genetics
R.P.V.
Master in Genomics and Genetics
Defense date
09.11.2024 10:30
09.11.2024 10:30
Summary
In woody species, and especially in recalcitrant species such as the holm oak, the in vitro morphogenetic capacity is generally higher in juvenile material than in adult material. The decline in morphogenetic capacity with plant age poses a limitation since trees do not exhibit the definitive characteristics that make them desirable for a specific use until they reach adulthood. This study aims to determine whether complete rejuvenation of adult holm oaks can be achieved through micropropagation via somatic embryogenesis (and its relationship with methylation). To this end, axillary shoot cultures were established from shoots obtained from forced sprouting of branches derived from two centennial holm oaks and one 30-year-old tree. For each of the three genotypes, apices and leaves isolated from these axillary cultures were used to establish two lines of micropropagated material, one from the axillary buds of the adult plant and another from somatic seedlings. Material from these two lines, as well as from axillary buds used as a control explant, were used to analyze DNA methylation markers (MSAP; Methylation-Sensitive Amplified Polymorphisms) to evaluate the possible involvement of epigenetic regulation in the different morphogenic and embryogenic capacities of adult material and rejuvenated material via somatic embryogenesis. The results indicated that the tissue source significantly influences DNA methylation patterns, with embryogenic seedlings showing different fragment frequencies compared to axillary buds. Finally, multilocus analyses indicated significant differences in methylation patterns between populations, influenced by both the age of the tree and the micropropagation technique, highlighting the utility of DNA methylation as a marker for studying epigenetic variability in these populations. However, the MSAP technique used to estimate this epigenetic differentiation showed a pattern of size homoplasy and a possible underestimation of methylation, suggesting the need for complementary methods to verify band homology.
In woody species, and especially in recalcitrant species such as the holm oak, the in vitro morphogenetic capacity is generally higher in juvenile material than in adult material. The decline in morphogenetic capacity with plant age poses a limitation since trees do not exhibit the definitive characteristics that make them desirable for a specific use until they reach adulthood. This study aims to determine whether complete rejuvenation of adult holm oaks can be achieved through micropropagation via somatic embryogenesis (and its relationship with methylation). To this end, axillary shoot cultures were established from shoots obtained from forced sprouting of branches derived from two centennial holm oaks and one 30-year-old tree. For each of the three genotypes, apices and leaves isolated from these axillary cultures were used to establish two lines of micropropagated material, one from the axillary buds of the adult plant and another from somatic seedlings. Material from these two lines, as well as from axillary buds used as a control explant, were used to analyze DNA methylation markers (MSAP; Methylation-Sensitive Amplified Polymorphisms) to evaluate the possible involvement of epigenetic regulation in the different morphogenic and embryogenic capacities of adult material and rejuvenated material via somatic embryogenesis. The results indicated that the tissue source significantly influences DNA methylation patterns, with embryogenic seedlings showing different fragment frequencies compared to axillary buds. Finally, multilocus analyses indicated significant differences in methylation patterns between populations, influenced by both the age of the tree and the micropropagation technique, highlighting the utility of DNA methylation as a marker for studying epigenetic variability in these populations. However, the MSAP technique used to estimate this epigenetic differentiation showed a pattern of size homoplasy and a possible underestimation of methylation, suggesting the need for complementary methods to verify band homology.
Direction
FERRADAS RIAL, YOLANDA (Tutorships)
FERRADAS RIAL, YOLANDA (Tutorships)
Court
Bouza Fernandez, M Carmen (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
DÍAZ FERNÁNDEZ, PABLO (Member)
Bouza Fernandez, M Carmen (Chairman)
VERA RODRIGUEZ, MANUEL (Secretary)
DÍAZ FERNÁNDEZ, PABLO (Member)