Construction of expression vectors for fluorescent proteins with specific subcellular localization.
Authorship
I.A.P.
Biotechnology Degree (2nd Ed. )
I.A.P.
Biotechnology Degree (2nd Ed. )
Defense date
07.18.2025 10:00
07.18.2025 10:00
Summary
The visualization of subcellular dynamics is essential for numerous studies in cell biology. One of the most commonly used approaches is the expression of fluorescent proteins such as enhanced green fluorescent protein (eGFP), enhanced yellow fluorescent protein (eYFP), Cerulean, or the red fluorescent protein from Discosoma sp. (DsRed), which allow tracking intracellular distribution in real time through fluorescence microscopy. To target these proteins to specific compartments, localization signals such as mitochondrial targeting sequences (MTS), nuclear export signals (NES), or nuclear localization signals (NLS) are used. This work aimed to construct a library of expression vectors in Saccharomyces cerevisiae for fluorescent proteins with defined subcellular localization, as well as to validate their expression using fluorescence microscopy. Gateway cloning techniques, vector purification, bacterial and yeast transformations, and microscopy analysis were employed. The results indicate that, among the proteins analyzed, eGFP showed the clearest signal. Cerulean and DsRed displayed signals that were difficult to distinguish from autofluorescence, making them indistinguishable from the cellular background. Regarding localization signals, eGFP showed expression consistent with mitochondrial targeting and nuclear export signals, while the nuclear localization sequence underwent a mutation that prevented its analysis. The development of a functional library of fluorescent vectors will enable studies on colocalization and subcellular dynamics in yeast, contributing to the functional analysis of proteins and the understanding of complex cellular processes.
The visualization of subcellular dynamics is essential for numerous studies in cell biology. One of the most commonly used approaches is the expression of fluorescent proteins such as enhanced green fluorescent protein (eGFP), enhanced yellow fluorescent protein (eYFP), Cerulean, or the red fluorescent protein from Discosoma sp. (DsRed), which allow tracking intracellular distribution in real time through fluorescence microscopy. To target these proteins to specific compartments, localization signals such as mitochondrial targeting sequences (MTS), nuclear export signals (NES), or nuclear localization signals (NLS) are used. This work aimed to construct a library of expression vectors in Saccharomyces cerevisiae for fluorescent proteins with defined subcellular localization, as well as to validate their expression using fluorescence microscopy. Gateway cloning techniques, vector purification, bacterial and yeast transformations, and microscopy analysis were employed. The results indicate that, among the proteins analyzed, eGFP showed the clearest signal. Cerulean and DsRed displayed signals that were difficult to distinguish from autofluorescence, making them indistinguishable from the cellular background. Regarding localization signals, eGFP showed expression consistent with mitochondrial targeting and nuclear export signals, while the nuclear localization sequence underwent a mutation that prevented its analysis. The development of a functional library of fluorescent vectors will enable studies on colocalization and subcellular dynamics in yeast, contributing to the functional analysis of proteins and the understanding of complex cellular processes.
Direction
González Blanco, Miguel (Tutorships)
González Blanco, Miguel (Tutorships)
Court
MARTINEZ COSTAS, JOSE MANUEL (Chairman)
GARCIA FANDIÑO, REBECA (Secretary)
CSABA , NOEMI STEFANIA (Member)
MARTINEZ COSTAS, JOSE MANUEL (Chairman)
GARCIA FANDIÑO, REBECA (Secretary)
CSABA , NOEMI STEFANIA (Member)
Development of functionalised biomimetic nanocapsules for the study of internalisation and intracellular routes of nanoparticles
Authorship
D.C.N.
Biotechnology Degree (2nd Ed. )
D.C.N.
Biotechnology Degree (2nd Ed. )
Defense date
07.18.2025 09:00
07.18.2025 09:00
Summary
The world of nanotechnology is a developing sector of great interest due to the great advances it can bring to many different fields. In the nanobiotechnology sector, nanoparticles stand out as vectors for delivering drugs in various therapies. For this project, the focus is on biomimetic nanosystems as vectors with unique properties and especially useful in delivering their cargo to cells. Thus, biomimetic nanovesicles based on the cell membrane of A549 cells, referred to as cellsomes, were developed, and gold nanoparticles with different degrees of ubiquitination were encapsulated in them in order to modify the cellular internalisation of the nanoparticles and to study their intracellular localisation. The use of electron microscopy, spectroscopy and nanoparticle tracking analysis techniques allowed us to characterise the cellsomes obtained, while the use of flow cytometry and confocal microscopy allowed us to study the internalisation of the nanoparticles and cellsomes as well as their localisation inside the cells. The results obtained show that synthesis and characterisation are feasible and successful, and that gold nanoparticles manage to enter both cellsomes and cells. Moreover, ubiquitination seems to help in the uptake of the nanoparticles, and the encapsulation of the nanoparticles in the cellsomes is able to change their intracellular localisation.
The world of nanotechnology is a developing sector of great interest due to the great advances it can bring to many different fields. In the nanobiotechnology sector, nanoparticles stand out as vectors for delivering drugs in various therapies. For this project, the focus is on biomimetic nanosystems as vectors with unique properties and especially useful in delivering their cargo to cells. Thus, biomimetic nanovesicles based on the cell membrane of A549 cells, referred to as cellsomes, were developed, and gold nanoparticles with different degrees of ubiquitination were encapsulated in them in order to modify the cellular internalisation of the nanoparticles and to study their intracellular localisation. The use of electron microscopy, spectroscopy and nanoparticle tracking analysis techniques allowed us to characterise the cellsomes obtained, while the use of flow cytometry and confocal microscopy allowed us to study the internalisation of the nanoparticles and cellsomes as well as their localisation inside the cells. The results obtained show that synthesis and characterisation are feasible and successful, and that gold nanoparticles manage to enter both cellsomes and cells. Moreover, ubiquitination seems to help in the uptake of the nanoparticles, and the encapsulation of the nanoparticles in the cellsomes is able to change their intracellular localisation.
Direction
DEL PINO GONZALEZ DE LA HIGUERA, PABLO ALFONSO (Tutorships)
PELAZ GARCIA, BEATRIZ (Co-tutorships)
DEL PINO GONZALEZ DE LA HIGUERA, PABLO ALFONSO (Tutorships)
PELAZ GARCIA, BEATRIZ (Co-tutorships)
Court
CID FERNANDEZ, MARIA MAGDALENA (Chairman)
LÓPEZ FABAL, ADOLFO (Secretary)
HOSPIDO QUINTANA, ALMUDENA (Member)
CID FERNANDEZ, MARIA MAGDALENA (Chairman)
LÓPEZ FABAL, ADOLFO (Secretary)
HOSPIDO QUINTANA, ALMUDENA (Member)
Biochemical, Molecular and Functional Characterization of a 3D Model Derived from Epicardial Mesenchymal Cells
Authorship
M.C.T.
Bachelor of Biology
M.C.T.
Bachelor of Biology
Defense date
07.18.2025 10:00
07.18.2025 10:00
Summary
Metabolic alterations derived from obesity contribute decisively to the development of cardiovascular diseases. In this context, epicardial adipose tissue stands out for its active role in cardiac dysfunction through the secretion of paracrine, endocrine, and vasocrine signals that affect both the myocardium and the coronary vasculature. Both epicardial and subcutaneous adipose tissues contain mesenchymal cells with a marked potential to induce adipogenesis and angiogenesis processes involved in cardiac remodeling and disease progression. Based on the hypothesis that a three-dimensional (3D) system would better reproduce the interaction between these processes, a 3D cell culture model was designed and characterized using mesenchymal cells isolated from subcutaneous and epicardial adipose tissue of patients undergoing cardiac surgery. Different extracellular matrices were evaluated, and specific treatments were applied to induce differentiation processes. The morphological and functional characterization of the model was carried out using optical microscopy, immunofluorescence, gene expression analysis by real-time PCR, and protein analysis by Western blot. The results confirmed the model’s capacity to reproduce adipogenesis and angiogenesis processes in an integrated manner, evidencing differences depending on the tissue origin. Subcutaneous adipose tissue showed a greater adipogenic capacity and three-dimensional organization, while epicardial adipose tissue exhibited a more inflammatory profile and a less cohesive structure. The angiogenic response was modest in both cases. Furthermore, structural and functional differences were observed between matrices, with Matrigel standing out as the most effective in forming stable spheroids. This model represents a useful preclinical tool to study the interaction between adipogenesis and angiogenesis processes in different tissues, as well as to explore new therapeutic approaches for cardiovascular diseases.
Metabolic alterations derived from obesity contribute decisively to the development of cardiovascular diseases. In this context, epicardial adipose tissue stands out for its active role in cardiac dysfunction through the secretion of paracrine, endocrine, and vasocrine signals that affect both the myocardium and the coronary vasculature. Both epicardial and subcutaneous adipose tissues contain mesenchymal cells with a marked potential to induce adipogenesis and angiogenesis processes involved in cardiac remodeling and disease progression. Based on the hypothesis that a three-dimensional (3D) system would better reproduce the interaction between these processes, a 3D cell culture model was designed and characterized using mesenchymal cells isolated from subcutaneous and epicardial adipose tissue of patients undergoing cardiac surgery. Different extracellular matrices were evaluated, and specific treatments were applied to induce differentiation processes. The morphological and functional characterization of the model was carried out using optical microscopy, immunofluorescence, gene expression analysis by real-time PCR, and protein analysis by Western blot. The results confirmed the model’s capacity to reproduce adipogenesis and angiogenesis processes in an integrated manner, evidencing differences depending on the tissue origin. Subcutaneous adipose tissue showed a greater adipogenic capacity and three-dimensional organization, while epicardial adipose tissue exhibited a more inflammatory profile and a less cohesive structure. The angiogenic response was modest in both cases. Furthermore, structural and functional differences were observed between matrices, with Matrigel standing out as the most effective in forming stable spheroids. This model represents a useful preclinical tool to study the interaction between adipogenesis and angiogenesis processes in different tissues, as well as to explore new therapeutic approaches for cardiovascular diseases.
Direction
BARREIRO IGLESIAS, ANTON (Tutorships)
Eiras Penas, Sonia (Co-tutorships)
BARREIRO IGLESIAS, ANTON (Tutorships)
Eiras Penas, Sonia (Co-tutorships)
Court
LAMAS FERNANDEZ, JESUS (Chairman)
DUBERT PEREZ, JAVIER (Secretary)
González Blanco, Miguel (Member)
LAMAS FERNANDEZ, JESUS (Chairman)
DUBERT PEREZ, JAVIER (Secretary)
González Blanco, Miguel (Member)
Metabolic Effect of Central Administration of a Pyk2 Inhibitor in Obese Rodents
Authorship
M.F.A.
Biotechnology Degree (2nd Ed. )
M.F.A.
Biotechnology Degree (2nd Ed. )
Defense date
07.18.2025 10:00
07.18.2025 10:00
Summary
In recent decades, obesity has become a serious public health problem worldwide, making the identification of new targets that could lead to novel therapeutic treatments critically important. Previous studies have determined that Pyk2 is involved in various diseases in which symptoms are associated with disruptions in energy balance. However, its role in obesity has not been thoroughly investigated. Therefore, the aim of this study is to examine the effect of central administration of a Pyk2 inhibitor on energy balance in obese mice. Our results show a reduction in body weight in these mice, which is explained by a decrease in food intake following intracerebroventricular administration of a Pyk2 inhibitor. In white adipose tissue and the liver, lipid metabolism and B-oxidation pathways exhibit a strong compensatory mechanism in response to weight loss. However, at the molecular level, an increase in Badr3 receptor expression was detected in white adipose tissue, along with a decrease in PPARg expression in the liver. Nonetheless, chronic administration of the Pyk2 inhibitor does not induce an increase in thermogenesis markers in BAT. Altogether, our findings provide the first experimental evidence pointing to a key role of Pyk2 in the central nervous system in the regulation of energy balance.
In recent decades, obesity has become a serious public health problem worldwide, making the identification of new targets that could lead to novel therapeutic treatments critically important. Previous studies have determined that Pyk2 is involved in various diseases in which symptoms are associated with disruptions in energy balance. However, its role in obesity has not been thoroughly investigated. Therefore, the aim of this study is to examine the effect of central administration of a Pyk2 inhibitor on energy balance in obese mice. Our results show a reduction in body weight in these mice, which is explained by a decrease in food intake following intracerebroventricular administration of a Pyk2 inhibitor. In white adipose tissue and the liver, lipid metabolism and B-oxidation pathways exhibit a strong compensatory mechanism in response to weight loss. However, at the molecular level, an increase in Badr3 receptor expression was detected in white adipose tissue, along with a decrease in PPARg expression in the liver. Nonetheless, chronic administration of the Pyk2 inhibitor does not induce an increase in thermogenesis markers in BAT. Altogether, our findings provide the first experimental evidence pointing to a key role of Pyk2 in the central nervous system in the regulation of energy balance.
Direction
QUIÑONES TELLEZ, MARIA DEL MAR (Tutorships)
AL-MASSADI IGLESIAS, OMAR (Co-tutorships)
QUIÑONES TELLEZ, MARIA DEL MAR (Tutorships)
AL-MASSADI IGLESIAS, OMAR (Co-tutorships)
Court
MARTINEZ COSTAS, JOSE MANUEL (Chairman)
GARCIA FANDIÑO, REBECA (Secretary)
CSABA , NOEMI STEFANIA (Member)
MARTINEZ COSTAS, JOSE MANUEL (Chairman)
GARCIA FANDIÑO, REBECA (Secretary)
CSABA , NOEMI STEFANIA (Member)
Role of lncRNAs and RNA binding proteins (RBPs) in liver disease
Authorship
H.F.H.
Bachelor of Biology
H.F.H.
Bachelor of Biology
Defense date
07.18.2025 10:00
07.18.2025 10:00
Summary
Chronic liver disease generates a fibrogenic response in the liver as a result of hepatic stellate cell activation, resulting in an accumulation of extracellular matrix in the liver as a mechanism for repairing damaged tissue. This process is highly regulated at multiple levels; one of the least studied regulatory mechanisms is that exerted by some lncRNAs in the activation of hepatic stellate cells. This final degree project explores the functional role of LncRNA-C1 in the development of liver fibrosis, with the aim of assessing its potential use in therapies for chronic liver disease. With this objective in mind, the levels of marker proteins of hepatic stellate cell activation and liver fibrosis, such as collagen and alpha-SMA, are analysed using histological techniques and Western blotting in two mouse models, bile duct ligation, which mimics human hepatic cholestasis, and a methionine-restricted, choline-deficient diet, which mimics metabolic associated fatty liver disease in mice in which the LncRNA-C1 gene has been deleted. Previously, using transient gene silencing models, the group had demonstrated the involvement of the LncRNA-C1 gene in the development of liver damage and fibrosis in the murine model of cholestasis. In this study, we observed increased activation of hepatic stellate cells in vivo after bile duct ligation in mice lacking the LncRNA-C1 gene, using histological techniques, when this damage extends over a period of more than 20 days. Possible improvements to the protocols, as well as new models capable of producing greater chronic damage, are proposed at the end of this study to study the impact of the absence of LncRNA-C1 on the development of chronic liver disease.
Chronic liver disease generates a fibrogenic response in the liver as a result of hepatic stellate cell activation, resulting in an accumulation of extracellular matrix in the liver as a mechanism for repairing damaged tissue. This process is highly regulated at multiple levels; one of the least studied regulatory mechanisms is that exerted by some lncRNAs in the activation of hepatic stellate cells. This final degree project explores the functional role of LncRNA-C1 in the development of liver fibrosis, with the aim of assessing its potential use in therapies for chronic liver disease. With this objective in mind, the levels of marker proteins of hepatic stellate cell activation and liver fibrosis, such as collagen and alpha-SMA, are analysed using histological techniques and Western blotting in two mouse models, bile duct ligation, which mimics human hepatic cholestasis, and a methionine-restricted, choline-deficient diet, which mimics metabolic associated fatty liver disease in mice in which the LncRNA-C1 gene has been deleted. Previously, using transient gene silencing models, the group had demonstrated the involvement of the LncRNA-C1 gene in the development of liver damage and fibrosis in the murine model of cholestasis. In this study, we observed increased activation of hepatic stellate cells in vivo after bile duct ligation in mice lacking the LncRNA-C1 gene, using histological techniques, when this damage extends over a period of more than 20 days. Possible improvements to the protocols, as well as new models capable of producing greater chronic damage, are proposed at the end of this study to study the impact of the absence of LncRNA-C1 on the development of chronic liver disease.
Direction
VARELA REY, MARTA MARIA (Tutorships)
VARELA REY, MARTA MARIA (Tutorships)
Court
LAMAS FERNANDEZ, JESUS (Chairman)
DUBERT PEREZ, JAVIER (Secretary)
González Blanco, Miguel (Member)
LAMAS FERNANDEZ, JESUS (Chairman)
DUBERT PEREZ, JAVIER (Secretary)
González Blanco, Miguel (Member)
Effect of partial chemical reprogramming on progeria
Authorship
B.F.M.
Biotechnology Degree (2nd Ed. )
B.F.M.
Biotechnology Degree (2nd Ed. )
Defense date
07.18.2025 10:00
07.18.2025 10:00
Summary
Hutchinson Gilford Progeria Syndrome is caused by the accumulation of farnesylated lamin A at the nuclear envelope, which leads to a prematurely aged phenotype in cells. This phenotype is identified by cell cycle arrest, abnormal nuclear morphology and senescence identity. Several strategies have been proposed to reverse cellular aging, focusing in partial reprogramming, among which the use of small chemical compounds such as tranylcypromine and RepSox stand out. These molecules act on an epigenetic level as well as on the transforming growth factor beta signalling pathway, thereby modifying gene expression and improving the aged phenotype. This study aims to evaluate the effectiveness of this chemical compounds in human dermal fibroblast that exhibit lamin A accumulation on the nuclear envelope. To do this, senescence and proliferative assays were performed to determine successful disease induction and subsequently to assess the effects of the proposes treatment. In addition, a genetic expression analysis is carried out to determine the effect of the treatment on epigenetic markers. Disease induction was successful, as demonstrated by clonogenic assays and microscopic observations. Moreover, treatment with tranylcypromine and RepSox proved beneficial, as evidenced by improvement in senescence markers, both genetic (CDKN1A, GDF-15 and SERPINE-1) and enzymatic (galactosidase activity) after a week of treatment.
Hutchinson Gilford Progeria Syndrome is caused by the accumulation of farnesylated lamin A at the nuclear envelope, which leads to a prematurely aged phenotype in cells. This phenotype is identified by cell cycle arrest, abnormal nuclear morphology and senescence identity. Several strategies have been proposed to reverse cellular aging, focusing in partial reprogramming, among which the use of small chemical compounds such as tranylcypromine and RepSox stand out. These molecules act on an epigenetic level as well as on the transforming growth factor beta signalling pathway, thereby modifying gene expression and improving the aged phenotype. This study aims to evaluate the effectiveness of this chemical compounds in human dermal fibroblast that exhibit lamin A accumulation on the nuclear envelope. To do this, senescence and proliferative assays were performed to determine successful disease induction and subsequently to assess the effects of the proposes treatment. In addition, a genetic expression analysis is carried out to determine the effect of the treatment on epigenetic markers. Disease induction was successful, as demonstrated by clonogenic assays and microscopic observations. Moreover, treatment with tranylcypromine and RepSox proved beneficial, as evidenced by improvement in senescence markers, both genetic (CDKN1A, GDF-15 and SERPINE-1) and enzymatic (galactosidase activity) after a week of treatment.
Direction
VIDAL FIGUEROA, ANXO (Tutorships)
Collado Rodríguez, Manuel (Co-tutorships)
DA SILVA ALVAREZ, SABELA (Co-tutorships)
VIDAL FIGUEROA, ANXO (Tutorships)
Collado Rodríguez, Manuel (Co-tutorships)
DA SILVA ALVAREZ, SABELA (Co-tutorships)
Court
MARTINEZ COSTAS, JOSE MANUEL (Chairman)
GARCIA FANDIÑO, REBECA (Secretary)
CSABA , NOEMI STEFANIA (Member)
MARTINEZ COSTAS, JOSE MANUEL (Chairman)
GARCIA FANDIÑO, REBECA (Secretary)
CSABA , NOEMI STEFANIA (Member)
Gene drives for pest control: a recap
Authorship
D.F.F.
Biotechnology Degree (2nd Ed. )
D.F.F.
Biotechnology Degree (2nd Ed. )
Defense date
07.18.2025 09:00
07.18.2025 09:00
Summary
Pest control remains one of the main challenges for public health, agriculture, and biodi-versity conservation. In recent decades, the development of genetic technologies such as gene drives has opened new possibilities for the specific and long-term control of tar-get species. This work aims to provide a bibliographic review and critical analysis of pest control methods based on gene drive technology, comparing them to conventional strat-egies and evaluating their environmental and social implications. The study examines the main molecular mechanisms employed, the types of gene drives developed to date, their technical limitations, and the ecological risks associated with their release. It also compares their advantages and drawbacks with traditional methods such as pesticides and identifies key ethical, regulatory, and scientific challenges that affect their future implementation. The results of this review suggest that although gene drives offer significant potential as a control tool, their use entails unresolved technical challenges and considerable ecolog-ical uncertainty. It is concluded that their application should proceed under robust inter-national governance frameworks, guided by scientific evidence, the precautionary princi-ple, and active public engagement.
Pest control remains one of the main challenges for public health, agriculture, and biodi-versity conservation. In recent decades, the development of genetic technologies such as gene drives has opened new possibilities for the specific and long-term control of tar-get species. This work aims to provide a bibliographic review and critical analysis of pest control methods based on gene drive technology, comparing them to conventional strat-egies and evaluating their environmental and social implications. The study examines the main molecular mechanisms employed, the types of gene drives developed to date, their technical limitations, and the ecological risks associated with their release. It also compares their advantages and drawbacks with traditional methods such as pesticides and identifies key ethical, regulatory, and scientific challenges that affect their future implementation. The results of this review suggest that although gene drives offer significant potential as a control tool, their use entails unresolved technical challenges and considerable ecolog-ical uncertainty. It is concluded that their application should proceed under robust inter-national governance frameworks, guided by scientific evidence, the precautionary princi-ple, and active public engagement.
Direction
GARCIA SUAREZ, CARLOS (Tutorships)
GARCIA SUAREZ, CARLOS (Tutorships)
Court
CID FERNANDEZ, MARIA MAGDALENA (Chairman)
LÓPEZ FABAL, ADOLFO (Secretary)
HOSPIDO QUINTANA, ALMUDENA (Member)
CID FERNANDEZ, MARIA MAGDALENA (Chairman)
LÓPEZ FABAL, ADOLFO (Secretary)
HOSPIDO QUINTANA, ALMUDENA (Member)
Water recovery and optimization in the wood industry
Authorship
A.G.B.
Biotechnology Degree (2nd Ed. )
A.G.B.
Biotechnology Degree (2nd Ed. )
Defense date
07.18.2025 09:00
07.18.2025 09:00
Summary
The scarcity of fresh water and environmental demands have driven the wood industry to look for more sustainable processes. This Final Degree Project was carried out at the Fibranor plant (Finsa group) and focused on studying the feasibility of recovering water in the gas scrubbing system (WESP) during the production of MDF boards. Mass and energy balances were carried out in three scenarios (current situation, gas cooling and condensation recovery), supported by psychrometric analysis and characterization of water samples from the system. The samples were analyzed to evaluate their possible reuse in the process. The results indicate that it is technically possible to recover up to 7,600 L/h of water. However, its high pollutant load requires a previous treatment, such as advanced oxidation, to be able to reuse it. This strategy would reduce water consumption and improve the plant's sustainability.
The scarcity of fresh water and environmental demands have driven the wood industry to look for more sustainable processes. This Final Degree Project was carried out at the Fibranor plant (Finsa group) and focused on studying the feasibility of recovering water in the gas scrubbing system (WESP) during the production of MDF boards. Mass and energy balances were carried out in three scenarios (current situation, gas cooling and condensation recovery), supported by psychrometric analysis and characterization of water samples from the system. The samples were analyzed to evaluate their possible reuse in the process. The results indicate that it is technically possible to recover up to 7,600 L/h of water. However, its high pollutant load requires a previous treatment, such as advanced oxidation, to be able to reuse it. This strategy would reduce water consumption and improve the plant's sustainability.
Direction
BALBOA MENDEZ, SABELA (Tutorships)
BALBOA MENDEZ, SABELA (Tutorships)
Court
CID FERNANDEZ, MARIA MAGDALENA (Chairman)
LÓPEZ FABAL, ADOLFO (Secretary)
HOSPIDO QUINTANA, ALMUDENA (Member)
CID FERNANDEZ, MARIA MAGDALENA (Chairman)
LÓPEZ FABAL, ADOLFO (Secretary)
HOSPIDO QUINTANA, ALMUDENA (Member)
Optimization of a Method for Purification and Sequencing of Long DNA Fragments from Low-Cellularity Biological Samples
Authorship
A.G.L.
Biotechnology Degree (2nd Ed. )
A.G.L.
Biotechnology Degree (2nd Ed. )
Defense date
07.18.2025 10:00
07.18.2025 10:00
Summary
Long-read sequencing offers advantages for the analysis of structural variants and repetitive regions of the human genome, but its application to samples with low DNA content requires optimized protocols. This study hypothesizes that an appropriate combination of preservation, lysis and amplification methods would yield DNA of sufficient quality for third-generation sequencing. To test this, three tissue-preservation methods (freezing at -80 degrees Celsius, PAXgene fixation without paraffin and paraffin embedding after PAXgene treatment) were compared, two lysis strategies (proteinase K versus the commercial REPLI-g Single Cell kit) were evaluated, and a whole-genome amplification and library-preparation workflow designed to preserve DNA structure was applied. The methodology combined fluorometric quantification, integrity assessment via TapeStation electropherograms, isothermal amplification, enzymatic debranching, magnetic purification and library preparation for subsequent sequencing. The combination of freezing or paraffin-free fixation with proteinase K lysis preserved high-molecular-weight DNA fragments and optimal integrity. The amplification and cleanup strategy efficiently retained enriched DNA, and the MinION run generated data with adequate coverage and quality to identify structural variants. These findings validate the proposed protocol for low-input samples and lay the groundwork for its application in clinical settings and in studies based on long-read technologies.
Long-read sequencing offers advantages for the analysis of structural variants and repetitive regions of the human genome, but its application to samples with low DNA content requires optimized protocols. This study hypothesizes that an appropriate combination of preservation, lysis and amplification methods would yield DNA of sufficient quality for third-generation sequencing. To test this, three tissue-preservation methods (freezing at -80 degrees Celsius, PAXgene fixation without paraffin and paraffin embedding after PAXgene treatment) were compared, two lysis strategies (proteinase K versus the commercial REPLI-g Single Cell kit) were evaluated, and a whole-genome amplification and library-preparation workflow designed to preserve DNA structure was applied. The methodology combined fluorometric quantification, integrity assessment via TapeStation electropherograms, isothermal amplification, enzymatic debranching, magnetic purification and library preparation for subsequent sequencing. The combination of freezing or paraffin-free fixation with proteinase K lysis preserved high-molecular-weight DNA fragments and optimal integrity. The amplification and cleanup strategy efficiently retained enriched DNA, and the MinION run generated data with adequate coverage and quality to identify structural variants. These findings validate the proposed protocol for low-input samples and lay the groundwork for its application in clinical settings and in studies based on long-read technologies.
Direction
CASTRO TUBIO, JOSE MANUEL (Tutorships)
RODRIGUEZ CASTRO, JORGE (Co-tutorships)
CASTRO TUBIO, JOSE MANUEL (Tutorships)
RODRIGUEZ CASTRO, JORGE (Co-tutorships)
Court
MARTINEZ COSTAS, JOSE MANUEL (Chairman)
GARCIA FANDIÑO, REBECA (Secretary)
CSABA , NOEMI STEFANIA (Member)
MARTINEZ COSTAS, JOSE MANUEL (Chairman)
GARCIA FANDIÑO, REBECA (Secretary)
CSABA , NOEMI STEFANIA (Member)
Evaluation of the effect of the ketone body beta-hydroxybutyrate on the methylome of human breast cancer cell lines.
Authorship
L.I.R.
Biotechnology Degree (2nd Ed. )
L.I.R.
Biotechnology Degree (2nd Ed. )
Defense date
07.18.2025 09:00
07.18.2025 09:00
Summary
Obesity is a chronic, complex, and multifactorial disease that has become a global public health problem and is associated with an increased risk of developing various comorbidities, including cancer. The most widely used method for diagnosis is the Body Mass Index (BMI), although more precise techniques exist to assess body composition. Among therapeutic strategies, the very-low-calorie ketogenic diet (VLCKD) has played a prominent role, promoting weight loss at the expense of fat mass and inducing a state of nutritional ketosis. Furthermore, obesity generates an inflammatory microenvironment and oxidative stress, caused by dysfunctional adipose tissue, as well as epigenetic alterations. These alterations have been proposed as possible mechanistic links between this condition and its associated diseases, such as cancer. It is hypothesized that ketone bodies could favorably interfere with tumor development by regulating epigenetic mechanisms, especially DNA methylation. Therefore, the main objective of this study was to evaluate in vitro how ketone bodies, specifically beta-hydroxybutyrate, affect the methylome of human breast cancer cell lines MCF-7 and MDA-MB-231. For this study, a bioinformatic and statistical analysis of the changes in the methylome of DNA samples derived from the treated breast cancer cell lines was performed. These samples were hybridized on the MethylationEPIC BeadChip Infinium (Illumina). 292,180 differentially methylated CpG sites were identified in MDA-MB-231 and 153,458 in MCF-7, with distinct epigenetic profiles between the two cell lines, indicating that ketone bodies can modulate DNA methylation in a cell subtype-specific manner, reinforcing the idea that they could play a beneficial effect on tumor development.
Obesity is a chronic, complex, and multifactorial disease that has become a global public health problem and is associated with an increased risk of developing various comorbidities, including cancer. The most widely used method for diagnosis is the Body Mass Index (BMI), although more precise techniques exist to assess body composition. Among therapeutic strategies, the very-low-calorie ketogenic diet (VLCKD) has played a prominent role, promoting weight loss at the expense of fat mass and inducing a state of nutritional ketosis. Furthermore, obesity generates an inflammatory microenvironment and oxidative stress, caused by dysfunctional adipose tissue, as well as epigenetic alterations. These alterations have been proposed as possible mechanistic links between this condition and its associated diseases, such as cancer. It is hypothesized that ketone bodies could favorably interfere with tumor development by regulating epigenetic mechanisms, especially DNA methylation. Therefore, the main objective of this study was to evaluate in vitro how ketone bodies, specifically beta-hydroxybutyrate, affect the methylome of human breast cancer cell lines MCF-7 and MDA-MB-231. For this study, a bioinformatic and statistical analysis of the changes in the methylome of DNA samples derived from the treated breast cancer cell lines was performed. These samples were hybridized on the MethylationEPIC BeadChip Infinium (Illumina). 292,180 differentially methylated CpG sites were identified in MDA-MB-231 and 153,458 in MCF-7, with distinct epigenetic profiles between the two cell lines, indicating that ketone bodies can modulate DNA methylation in a cell subtype-specific manner, reinforcing the idea that they could play a beneficial effect on tumor development.
Direction
VIDAL FIGUEROA, ANXO (Tutorships)
CRUJEIRAS MARTINEZ, ANA BELEN (Co-tutorships)
VIDAL FIGUEROA, ANXO (Tutorships)
CRUJEIRAS MARTINEZ, ANA BELEN (Co-tutorships)
Court
EIBES GONZALEZ, GEMMA MARIA (Chairman)
CASTRO TUBIO, JOSE MANUEL (Secretary)
GARCIA ALONSO, ANGEL (Member)
EIBES GONZALEZ, GEMMA MARIA (Chairman)
CASTRO TUBIO, JOSE MANUEL (Secretary)
GARCIA ALONSO, ANGEL (Member)
Effect of mepolizumab on the low-abundance serum proteome of patients with severe eosinophilic asthma.
Authorship
A.M.G.
Bachelor of Biology
A.M.G.
Bachelor of Biology
Defense date
07.18.2025 10:00
07.18.2025 10:00
Summary
Severe eosinophilic asthma (SEA) represents a subtype of the T2high phenotype characterized by persistent eosinophil-mediated inflammation and resistance to conventional treatment with inhaled corticosteroids. Interleukin 5 (IL-5) plays a key role in the activation, maturation and survival of eosinophils, making it a therapeutic target for biological drugs such as mepolizumab (an anti-IL5 antibody). This study evaluates the effect of mepolizumab treatment on the low-abundance serum proteome in patients with SEA, with samples collected from healthy controls and asthmatic patients at four time points: before treatment (T0) and at 4, 16, and 32 weeks after treatment initiation (T4, T16, T32). Samples were treated with DTT to remove the high-abundance proteome, and using mass spectrometry (LC-MS/MS) and the SWATH-MS technique, it was observed that healthy controls and patients at the end of treatment (T32) showed a similar profile. Similarly, the profiles before treatment (T0) and at 4 weeks (T4) were also alike. At T0, the proteins APOH and PROS1, related to coagulation, and SERPINA1 and SERPINA3, related to the acute phase response, were found to be overexpressed. These, along with SERPINA6, which was also overexpressed, showed high predictive power (AUC higher than 0.8), with the latter possibly having the greatest implication in the differences between healthy controls and T0, and between T0 and T32.
Severe eosinophilic asthma (SEA) represents a subtype of the T2high phenotype characterized by persistent eosinophil-mediated inflammation and resistance to conventional treatment with inhaled corticosteroids. Interleukin 5 (IL-5) plays a key role in the activation, maturation and survival of eosinophils, making it a therapeutic target for biological drugs such as mepolizumab (an anti-IL5 antibody). This study evaluates the effect of mepolizumab treatment on the low-abundance serum proteome in patients with SEA, with samples collected from healthy controls and asthmatic patients at four time points: before treatment (T0) and at 4, 16, and 32 weeks after treatment initiation (T4, T16, T32). Samples were treated with DTT to remove the high-abundance proteome, and using mass spectrometry (LC-MS/MS) and the SWATH-MS technique, it was observed that healthy controls and patients at the end of treatment (T32) showed a similar profile. Similarly, the profiles before treatment (T0) and at 4 weeks (T4) were also alike. At T0, the proteins APOH and PROS1, related to coagulation, and SERPINA1 and SERPINA3, related to the acute phase response, were found to be overexpressed. These, along with SERPINA6, which was also overexpressed, showed high predictive power (AUC higher than 0.8), with the latter possibly having the greatest implication in the differences between healthy controls and T0, and between T0 and T32.
Direction
NIETO FONTARIGO, JUAN JOSE (Tutorships)
NIETO FONTARIGO, JUAN JOSE (Tutorships)
Court
LAMAS FERNANDEZ, JESUS (Chairman)
DUBERT PEREZ, JAVIER (Secretary)
González Blanco, Miguel (Member)
LAMAS FERNANDEZ, JESUS (Chairman)
DUBERT PEREZ, JAVIER (Secretary)
González Blanco, Miguel (Member)
Determination of potential genetic interactions between deregulated versions of YEN1 and genes involved in the removal of DNA protein adducts
Authorship
L.R.F.
Biotechnology Degree (2nd Ed. )
L.R.F.
Biotechnology Degree (2nd Ed. )
Defense date
07.18.2025 09:00
07.18.2025 09:00
Summary
The maintenance of genome stability relies on a broad set of DNA repair mechanisms, among which structure-specific nucleases play a key role. In Saccharomyces cerevisiae, the endonuclease Yen1 performs an essential function as a Holliday junction resolvase, and its activity is tightly regulated throughout the cell cycle. Several studies have shown that deregulated versions of Yen1- either constitutively expressed or phosphomutant-can be toxic if they access DNA prematurely during S phase. In this context, the expression of a catalytically inactive variant (Yen1ON-ND) retains such toxicity, suggesting the formation of protein-DNA crosslinks (DPCs), a highly obstructive type of lesion that interferes with both replication and transcription. This Bachelor’s Thesis investigates whether the overexpression of factors involved in DPC resolution can suppress such toxicity. To this end, the genes WSS1, CDC48, and DDI1 were cloned using the Gateway system and constitutively expressed in S. cerevisiae. WSS1 encodes a DNA-dependent protease that acts on DPCs in cooperation with the ATPase Cdc48, which segregates SUMO- or ubiquitin-modified proteins from chromatin. DDI1, in turn, may function redundantly when the main repair pathways fail. Although the interaction between these proteins and Yen1ON-ND could not be confirmed, the results suggest that WSS1 itself may be toxic under the conditions tested. This work lays the foundation for future studies to explore the suppressive potential of WSS1, CDC48, and DDI1 under alternative inducible promoters.
The maintenance of genome stability relies on a broad set of DNA repair mechanisms, among which structure-specific nucleases play a key role. In Saccharomyces cerevisiae, the endonuclease Yen1 performs an essential function as a Holliday junction resolvase, and its activity is tightly regulated throughout the cell cycle. Several studies have shown that deregulated versions of Yen1- either constitutively expressed or phosphomutant-can be toxic if they access DNA prematurely during S phase. In this context, the expression of a catalytically inactive variant (Yen1ON-ND) retains such toxicity, suggesting the formation of protein-DNA crosslinks (DPCs), a highly obstructive type of lesion that interferes with both replication and transcription. This Bachelor’s Thesis investigates whether the overexpression of factors involved in DPC resolution can suppress such toxicity. To this end, the genes WSS1, CDC48, and DDI1 were cloned using the Gateway system and constitutively expressed in S. cerevisiae. WSS1 encodes a DNA-dependent protease that acts on DPCs in cooperation with the ATPase Cdc48, which segregates SUMO- or ubiquitin-modified proteins from chromatin. DDI1, in turn, may function redundantly when the main repair pathways fail. Although the interaction between these proteins and Yen1ON-ND could not be confirmed, the results suggest that WSS1 itself may be toxic under the conditions tested. This work lays the foundation for future studies to explore the suppressive potential of WSS1, CDC48, and DDI1 under alternative inducible promoters.
Direction
González Blanco, Miguel (Tutorships)
González Blanco, Miguel (Tutorships)
Court
EIBES GONZALEZ, GEMMA MARIA (Chairman)
CASTRO TUBIO, JOSE MANUEL (Secretary)
GARCIA ALONSO, ANGEL (Member)
EIBES GONZALEZ, GEMMA MARIA (Chairman)
CASTRO TUBIO, JOSE MANUEL (Secretary)
GARCIA ALONSO, ANGEL (Member)
Development of targeted systems for apoptosis induction
Authorship
A.R.R.
Biotechnology Degree (2nd Ed. )
A.R.R.
Biotechnology Degree (2nd Ed. )
Defense date
07.18.2025 09:00
07.18.2025 09:00
Summary
Intercellular communication plays a key role in the response to tissue damage. The regulation of processes such as programmed cell death and senescence, commonly considered independent mechanisms, is essential for the proper repair and regeneration of damaged tissue. In this context, recent studies in our laboratory have suggested that certain forms of cell death could induce senescence in neighboring cells through paracrine signals. This study aimed to explore this hypothesis using a mitochondria-independent, controlled apoptosis system based on the induced activation of caspase 9. To this end, a TC1 cell line modified to express an inducible version of caspase 9 (iCasp9-GFP), activated by the dimerizing compound AP20187, was generated. The apoptotic cells were exposed to primary murine embryonic fibroblasts using in vitro cultures with conditioned media, transwell systems, and direct co-culture, after which the potential induction of senescence was evaluated. In all cases, the results showed that apoptosis induction was not sufficient to paracrine-activate a senescent phenotype in the target cells, in contrast to positive controls of cells induced to senescence by treatment with traditional chemotherapeutics. These findings allow us to separate the cell death process from the accompanying signaling, suggesting that this signaling (and not apoptosis itself) is responsible for inducing paracrine senescence, with other factors such as mitochondrial dysfunction or the immunological context being possible triggers. The study provides a useful framework for further exploring the complex interplay between cell death and senescence, with potential therapeutic implications for diseases associated with aging or tissue regeneration.
Intercellular communication plays a key role in the response to tissue damage. The regulation of processes such as programmed cell death and senescence, commonly considered independent mechanisms, is essential for the proper repair and regeneration of damaged tissue. In this context, recent studies in our laboratory have suggested that certain forms of cell death could induce senescence in neighboring cells through paracrine signals. This study aimed to explore this hypothesis using a mitochondria-independent, controlled apoptosis system based on the induced activation of caspase 9. To this end, a TC1 cell line modified to express an inducible version of caspase 9 (iCasp9-GFP), activated by the dimerizing compound AP20187, was generated. The apoptotic cells were exposed to primary murine embryonic fibroblasts using in vitro cultures with conditioned media, transwell systems, and direct co-culture, after which the potential induction of senescence was evaluated. In all cases, the results showed that apoptosis induction was not sufficient to paracrine-activate a senescent phenotype in the target cells, in contrast to positive controls of cells induced to senescence by treatment with traditional chemotherapeutics. These findings allow us to separate the cell death process from the accompanying signaling, suggesting that this signaling (and not apoptosis itself) is responsible for inducing paracrine senescence, with other factors such as mitochondrial dysfunction or the immunological context being possible triggers. The study provides a useful framework for further exploring the complex interplay between cell death and senescence, with potential therapeutic implications for diseases associated with aging or tissue regeneration.
Direction
VIDAL FIGUEROA, ANXO (Tutorships)
Collado Rodríguez, Manuel (Co-tutorships)
DA SILVA ALVAREZ, SABELA (Co-tutorships)
VIDAL FIGUEROA, ANXO (Tutorships)
Collado Rodríguez, Manuel (Co-tutorships)
DA SILVA ALVAREZ, SABELA (Co-tutorships)
Court
CID FERNANDEZ, MARIA MAGDALENA (Chairman)
LÓPEZ FABAL, ADOLFO (Secretary)
HOSPIDO QUINTANA, ALMUDENA (Member)
CID FERNANDEZ, MARIA MAGDALENA (Chairman)
LÓPEZ FABAL, ADOLFO (Secretary)
HOSPIDO QUINTANA, ALMUDENA (Member)
Biorefinery design by modeling. Case study of brewer’s spent grain
Authorship
P.S.M.
Biotechnology Degree (2nd Ed. )
P.S.M.
Biotechnology Degree (2nd Ed. )
Defense date
07.18.2025 09:00
07.18.2025 09:00
Summary
This Bachelor's Thesis presents an approach to the preliminary design of a biorefinery, based on the definition and optimization of a superstructure. The case study focuses on brewer’s spent grain (BSG), the main by-product of the brewing industry. Through a bibliographic review, multiple valorization pathways were identified. The selected technologies, which were chosen in order to target the utilization of different fractions of BSG, were then characterized. These valorization alternatives were integrated into a superstructure, allowing the comparison of the various proposed processes for obtaining commercially valuable products. In this context, the use of BSG as a feedstock in microbially mediated transformations was assessed by applying genome-scale metabolic models (GEMs). These models enabled a screening of microorganisms reported in the literature, helping to identify potential carbon sources, possible products, and their respective yields. Finally, the superstructure optimization problem was formulated and solved using OUTDOOR, determining the most economically favorable pathway. Among the various solutions explored and compared, the most promising processes identified were: (1) direct composting of BSG, (2) extraction of phenolic compounds followed by anaerobic digestion of the remaining fraction, and (3) protein extraction combined with a biological transformation mediated by Saccharomyces cerevisiae. This methodology proved to be a robust, flexible, and replicable tool with strong potential for application in the early stages of biorefinery design aimed at the valorization of various agro-industrial residues.
This Bachelor's Thesis presents an approach to the preliminary design of a biorefinery, based on the definition and optimization of a superstructure. The case study focuses on brewer’s spent grain (BSG), the main by-product of the brewing industry. Through a bibliographic review, multiple valorization pathways were identified. The selected technologies, which were chosen in order to target the utilization of different fractions of BSG, were then characterized. These valorization alternatives were integrated into a superstructure, allowing the comparison of the various proposed processes for obtaining commercially valuable products. In this context, the use of BSG as a feedstock in microbially mediated transformations was assessed by applying genome-scale metabolic models (GEMs). These models enabled a screening of microorganisms reported in the literature, helping to identify potential carbon sources, possible products, and their respective yields. Finally, the superstructure optimization problem was formulated and solved using OUTDOOR, determining the most economically favorable pathway. Among the various solutions explored and compared, the most promising processes identified were: (1) direct composting of BSG, (2) extraction of phenolic compounds followed by anaerobic digestion of the remaining fraction, and (3) protein extraction combined with a biological transformation mediated by Saccharomyces cerevisiae. This methodology proved to be a robust, flexible, and replicable tool with strong potential for application in the early stages of biorefinery design aimed at the valorization of various agro-industrial residues.
Direction
MAURICIO IGLESIAS, MIGUEL (Tutorships)
MAURICIO IGLESIAS, MIGUEL (Tutorships)
Court
EIBES GONZALEZ, GEMMA MARIA (Chairman)
CASTRO TUBIO, JOSE MANUEL (Secretary)
GARCIA ALONSO, ANGEL (Member)
EIBES GONZALEZ, GEMMA MARIA (Chairman)
CASTRO TUBIO, JOSE MANUEL (Secretary)
GARCIA ALONSO, ANGEL (Member)
Impact of Somatic LINE-1 Integration on RNA Structure in Human Tumors
Authorship
L.S.P.
Biotechnology Degree (2nd Ed. )
L.S.P.
Biotechnology Degree (2nd Ed. )
Defense date
07.18.2025 09:00
07.18.2025 09:00
Summary
Transposable elements (TEs) are DNA sequences capable of changing their location within the genome that harbors them, a process known as transposition. TEs are classified into two main types based on their transposition mechanism: DNA transposons and RNA transposons, or retrotransposons. In mammals, they typically make up around 50% of the genome. As a final consequence of transposition, the integration of these sequences can cause genetic mutations, thereby influencing both species evolution and the development of diseases such as cancer. In fact, recent studies have shown that retrotransposition is highly active in certain human tumors. At the transcriptional level, it has been observed that retrotransposon insertions can alter RNA splicing or promote alternative transcription start or termination sites. However, RNA-level mutational mechanisms have been poorly studied, mainly due to limitations in short-read sequencing technologies (the most commonly used to date). This study aims to evaluate the impact of retrotransposon integration, primarily LINE-1 (L1) elements, on transcript structure in human tumors. To achieve this, we performed direct RNA sequencing using long-read technology, generating the transcriptomes of three human tumors with high rates of retrotransposon mobilization. We then analyzed these data using bioinformatic approaches to identify potential alterations in RNA processing. Our analysis identified somatic LINE-1 insertions associated with aberrant RNA structures, including retrotransposon exonization and premature transcription termination. These findings highlight the potential of somatic retrotransposon integration to impact gene expression and function in cancer, paving the way for future studies that further investigate these mutational mechanisms and their possible contribution to cancer development.
Transposable elements (TEs) are DNA sequences capable of changing their location within the genome that harbors them, a process known as transposition. TEs are classified into two main types based on their transposition mechanism: DNA transposons and RNA transposons, or retrotransposons. In mammals, they typically make up around 50% of the genome. As a final consequence of transposition, the integration of these sequences can cause genetic mutations, thereby influencing both species evolution and the development of diseases such as cancer. In fact, recent studies have shown that retrotransposition is highly active in certain human tumors. At the transcriptional level, it has been observed that retrotransposon insertions can alter RNA splicing or promote alternative transcription start or termination sites. However, RNA-level mutational mechanisms have been poorly studied, mainly due to limitations in short-read sequencing technologies (the most commonly used to date). This study aims to evaluate the impact of retrotransposon integration, primarily LINE-1 (L1) elements, on transcript structure in human tumors. To achieve this, we performed direct RNA sequencing using long-read technology, generating the transcriptomes of three human tumors with high rates of retrotransposon mobilization. We then analyzed these data using bioinformatic approaches to identify potential alterations in RNA processing. Our analysis identified somatic LINE-1 insertions associated with aberrant RNA structures, including retrotransposon exonization and premature transcription termination. These findings highlight the potential of somatic retrotransposon integration to impact gene expression and function in cancer, paving the way for future studies that further investigate these mutational mechanisms and their possible contribution to cancer development.
Direction
CASTRO TUBIO, JOSE MANUEL (Tutorships)
RODRIGUEZ CASTRO, JORGE (Co-tutorships)
CASTRO TUBIO, JOSE MANUEL (Tutorships)
RODRIGUEZ CASTRO, JORGE (Co-tutorships)
Court
CID FERNANDEZ, MARIA MAGDALENA (Chairman)
LÓPEZ FABAL, ADOLFO (Secretary)
HOSPIDO QUINTANA, ALMUDENA (Member)
CID FERNANDEZ, MARIA MAGDALENA (Chairman)
LÓPEZ FABAL, ADOLFO (Secretary)
HOSPIDO QUINTANA, ALMUDENA (Member)
Identification of lncRNAs involved in the development of liver disease
Authorship
P.S.G.
Biotechnology Degree (2nd Ed. )
P.S.G.
Biotechnology Degree (2nd Ed. )
Defense date
07.18.2025 09:00
07.18.2025 09:00
Summary
Hepatic fibrosis is a progressive scarring process that disrupts the architecture and function of the liver. Hepatic stellate cells (HSCs) and transforming growth factor-beta (TGF-ß) are key players in this progression. Based on the hypothesis that certain long non-coding RNAs may modulate this activation, this study aimed to analyze the transcriptional changes induced by TGF-ß through RNA-Seq, using libraries generated via rRNA depletion in the LX-2 hepatic stellate cell line. Quality control analysis revealed a technical issue in library preparation, which led to the development of a more stringent and context-specific filtering method. This was made possible by the availability of equivalent data generated from polyadenylated transcript selection libraries. Ultimately, 2,651 differentially expressed genes were identified, 521 of which correspond to long non-coding RNAs. Notable findings include the overexpression of the mitochondrial gene MT-ND6 and the regulatory gene GPRACR, which is associated with collagen synthesis. Additionally, downregulation of histone-coding genes involved in chromatin organization was observed. These results contribute to a deeper understanding of the mechanisms driving hepatic fibrosis and highlight new diagnostic and therapeutic targets.
Hepatic fibrosis is a progressive scarring process that disrupts the architecture and function of the liver. Hepatic stellate cells (HSCs) and transforming growth factor-beta (TGF-ß) are key players in this progression. Based on the hypothesis that certain long non-coding RNAs may modulate this activation, this study aimed to analyze the transcriptional changes induced by TGF-ß through RNA-Seq, using libraries generated via rRNA depletion in the LX-2 hepatic stellate cell line. Quality control analysis revealed a technical issue in library preparation, which led to the development of a more stringent and context-specific filtering method. This was made possible by the availability of equivalent data generated from polyadenylated transcript selection libraries. Ultimately, 2,651 differentially expressed genes were identified, 521 of which correspond to long non-coding RNAs. Notable findings include the overexpression of the mitochondrial gene MT-ND6 and the regulatory gene GPRACR, which is associated with collagen synthesis. Additionally, downregulation of histone-coding genes involved in chromatin organization was observed. These results contribute to a deeper understanding of the mechanisms driving hepatic fibrosis and highlight new diagnostic and therapeutic targets.
Direction
VARELA REY, MARTA MARIA (Tutorships)
ESQUINAS ROMAN, EVA MARIA (Co-tutorships)
VARELA REY, MARTA MARIA (Tutorships)
ESQUINAS ROMAN, EVA MARIA (Co-tutorships)
Court
EIBES GONZALEZ, GEMMA MARIA (Chairman)
CASTRO TUBIO, JOSE MANUEL (Secretary)
GARCIA ALONSO, ANGEL (Member)
EIBES GONZALEZ, GEMMA MARIA (Chairman)
CASTRO TUBIO, JOSE MANUEL (Secretary)
GARCIA ALONSO, ANGEL (Member)
Stress epigenetics: mechanisms and evidence of generational transmission
Authorship
J.V.C.
Bachelor of Biology
J.V.C.
Bachelor of Biology
Defense date
07.18.2025 10:00
07.18.2025 10:00
Summary
Environmental factors can induce epigenetic modifications that alter gene expression without changing the DNA sequence, with stress standing out due to its current impact on mental health. As long as these modifications escape the two reprogramming processes in the germline of each generation, they can affect both intergenerationally and transgenerationally, which could contribute to a predisposition to psychological disorders in future generations. In this way, the widespread acceptance that stress leaves an epigenetic mark affecting subsequent generations is explored, analyzing the available evidence, the molecular mechanisms involved (DNA methylation, histone modification, and non-coding RNA), and how they work, as well as the factors that influence their appearance and transmission. The literature review shows that exposure to environmental factors during windows of vulnerability, when the epigenome is especially sensitive, increases the likelihood that epigenetic modifications will affect the germline. However, while the reviewed studies support intergenerational epigenetic inheritance in both animal models and humans, only one experiment in rats provides direct molecular evidence of a transgenerational epigenetic transmission caused by stress.
Environmental factors can induce epigenetic modifications that alter gene expression without changing the DNA sequence, with stress standing out due to its current impact on mental health. As long as these modifications escape the two reprogramming processes in the germline of each generation, they can affect both intergenerationally and transgenerationally, which could contribute to a predisposition to psychological disorders in future generations. In this way, the widespread acceptance that stress leaves an epigenetic mark affecting subsequent generations is explored, analyzing the available evidence, the molecular mechanisms involved (DNA methylation, histone modification, and non-coding RNA), and how they work, as well as the factors that influence their appearance and transmission. The literature review shows that exposure to environmental factors during windows of vulnerability, when the epigenome is especially sensitive, increases the likelihood that epigenetic modifications will affect the germline. However, while the reviewed studies support intergenerational epigenetic inheritance in both animal models and humans, only one experiment in rats provides direct molecular evidence of a transgenerational epigenetic transmission caused by stress.
Direction
CANDAL SUAREZ, EVA MARIA (Tutorships)
CANDAL SUAREZ, EVA MARIA (Tutorships)
Court
LAMAS FERNANDEZ, JESUS (Chairman)
DUBERT PEREZ, JAVIER (Secretary)
González Blanco, Miguel (Member)
LAMAS FERNANDEZ, JESUS (Chairman)
DUBERT PEREZ, JAVIER (Secretary)
González Blanco, Miguel (Member)
Alterations in the blood-brain barrier (BBB) in cancer-associated cachexia
Authorship
L.V.V.
Biotechnology Degree (2nd Ed. )
L.V.V.
Biotechnology Degree (2nd Ed. )
Defense date
07.18.2025 10:00
07.18.2025 10:00
Summary
Cancer-associated cachexia is a multifactorial syndrome that alters the patient's metabolic state. The systemic inflammatory response induced by the tumour can affect the integrity of the blood-brain barrier (BBB), causing a dysregulation of the central nervous system that contributes to the progression of cachexia. Understanding the relationship between cancer-associated cachexia and the BBB could contribute to improving its clinical management and reducing its potential neurological complications. The hypothesis of this study is that a simple in vitro model of the BBB allows the study of alterations induced by circulating factors present in the serum of mice with cancer-induced cachexia. The aim of this study was to evaluate the effect of these sera on the structure of the BHE. To this end, an in vitro model of BHE based on bEnd.3 cells treated with sera from mice with Lewis lung carcinoma or control mouse sera was used. Cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), reactive oxygen species (ROS) production by flow cytometry, alterations in the endothelial barrier structure by determining transendothelial electrical resistance (TEER) and tight junction (TJ) protein expression by Western blot. The main results indicate that, at the concentration used, sera from mice with cachexia do not induce a significant decrease in cell viability, do not cause a significant increase in ROS production, but tend to reduce TEER after prolonged exposure of endothelial cells, without generating significant changes in tight junction protein expression. Therefore, the hypothesis could not be definitively validated, and studies with greater statistical power using different serum concentrations are needed to obtain more conclusive results.
Cancer-associated cachexia is a multifactorial syndrome that alters the patient's metabolic state. The systemic inflammatory response induced by the tumour can affect the integrity of the blood-brain barrier (BBB), causing a dysregulation of the central nervous system that contributes to the progression of cachexia. Understanding the relationship between cancer-associated cachexia and the BBB could contribute to improving its clinical management and reducing its potential neurological complications. The hypothesis of this study is that a simple in vitro model of the BBB allows the study of alterations induced by circulating factors present in the serum of mice with cancer-induced cachexia. The aim of this study was to evaluate the effect of these sera on the structure of the BHE. To this end, an in vitro model of BHE based on bEnd.3 cells treated with sera from mice with Lewis lung carcinoma or control mouse sera was used. Cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), reactive oxygen species (ROS) production by flow cytometry, alterations in the endothelial barrier structure by determining transendothelial electrical resistance (TEER) and tight junction (TJ) protein expression by Western blot. The main results indicate that, at the concentration used, sera from mice with cachexia do not induce a significant decrease in cell viability, do not cause a significant increase in ROS production, but tend to reduce TEER after prolonged exposure of endothelial cells, without generating significant changes in tight junction protein expression. Therefore, the hypothesis could not be definitively validated, and studies with greater statistical power using different serum concentrations are needed to obtain more conclusive results.
Direction
VIÑA CASTELAO, MARÍA DOLORES (Tutorships)
SEÑARIS RODRIGUEZ, ROSA MARIA (Co-tutorships)
VIÑA CASTELAO, MARÍA DOLORES (Tutorships)
SEÑARIS RODRIGUEZ, ROSA MARIA (Co-tutorships)
Court
MARTINEZ COSTAS, JOSE MANUEL (Chairman)
GARCIA FANDIÑO, REBECA (Secretary)
CSABA , NOEMI STEFANIA (Member)
MARTINEZ COSTAS, JOSE MANUEL (Chairman)
GARCIA FANDIÑO, REBECA (Secretary)
CSABA , NOEMI STEFANIA (Member)
Evaluation of the antiviral activity of different ionic liquids against the nervous necrosis virus (NNV) and the spring viremia of carp virus (SVCV)
Authorship
M.M.V.B.
Bachelor of Biology
M.M.V.B.
Bachelor of Biology
Defense date
07.18.2025 10:00
07.18.2025 10:00
Summary
Ionic liquids (ILs) are compounds composed exclusively of ions, with physicochemical properties that make them potentially useful in biomedical applications. In this study, the antiviral activity of two ionic liquids, Betaine TFSI and AMIM Cl, was evaluated against two viruses relevant to aquaculture: the nervous necrosis virus (NNV), a non-enveloped virus, and the spring viremia of carp virus (SVCV), an enveloped virus. Cytotoxicity assays were conducted on specific cell lines, along with tests to assess inhibition of viral adsorption and replication. The findings demonstrate that certain ionic liquids have the capacity to interfere with the viral replication cycle, suggesting their potential as innovative tools for controlling infectious diseases in aquaculture.
Ionic liquids (ILs) are compounds composed exclusively of ions, with physicochemical properties that make them potentially useful in biomedical applications. In this study, the antiviral activity of two ionic liquids, Betaine TFSI and AMIM Cl, was evaluated against two viruses relevant to aquaculture: the nervous necrosis virus (NNV), a non-enveloped virus, and the spring viremia of carp virus (SVCV), an enveloped virus. Cytotoxicity assays were conducted on specific cell lines, along with tests to assess inhibition of viral adsorption and replication. The findings demonstrate that certain ionic liquids have the capacity to interfere with the viral replication cycle, suggesting their potential as innovative tools for controlling infectious diseases in aquaculture.
Direction
BANDIN MATOS, MARIA ISABEL (Tutorships)
SOUTO PEREIRA, SANDRA (Co-tutorships)
BANDIN MATOS, MARIA ISABEL (Tutorships)
SOUTO PEREIRA, SANDRA (Co-tutorships)
Court
LAMAS FERNANDEZ, JESUS (Chairman)
DUBERT PEREZ, JAVIER (Secretary)
González Blanco, Miguel (Member)
LAMAS FERNANDEZ, JESUS (Chairman)
DUBERT PEREZ, JAVIER (Secretary)
González Blanco, Miguel (Member)