Development of a miniaturized phenotypic assay for drug screening in polycystic kidney disease
Authorship
J.A.M.
Master in Drug Research and Development
J.A.M.
Master in Drug Research and Development
Defense date
07.14.2025 10:00
07.14.2025 10:00
Summary
Polycystic kidney disease, particularly the autosomal dominant form (ADPKD), is a genetic disorder characterized by the formation of cysts derived from the renal tubular epithelium. Approximately half of patients require dialysis or kidney replacement therapy before the age of 60 due to the limited efficacy and significant side effects of tolvaptan, the only approved drug for this condition. Current disease models have remarkable limitations and are mainly used in target-based drug discovery strategies, thereby producing a bias towards already known molecular targets and limiting identification of novel mechanisms. Therefore, developing models with a phenotypic approach focused on cellular response is essential and may allow the identification of new treatments. The objective of this study was to develop a miniaturized phenotypic assay using immortalized MDCK (Madin-Darby Canine Kidney) cells, with potential applicability in high-throughput screening (HTS). To this end, we established culture conditions in a collagen matrix, employed forskolin to induce cystogenesis, and tested the effects of rapamycin and birinapant. Forskolin induced cyst formation at different concentrations, and both rapamycin and birinapant reduced cyst number. In conclusion, we successfully developed a miniaturized 3D phenotypic ADPKD model in 384-well plates, with potential applicability HTS-based drug discovery in this disease.
Polycystic kidney disease, particularly the autosomal dominant form (ADPKD), is a genetic disorder characterized by the formation of cysts derived from the renal tubular epithelium. Approximately half of patients require dialysis or kidney replacement therapy before the age of 60 due to the limited efficacy and significant side effects of tolvaptan, the only approved drug for this condition. Current disease models have remarkable limitations and are mainly used in target-based drug discovery strategies, thereby producing a bias towards already known molecular targets and limiting identification of novel mechanisms. Therefore, developing models with a phenotypic approach focused on cellular response is essential and may allow the identification of new treatments. The objective of this study was to develop a miniaturized phenotypic assay using immortalized MDCK (Madin-Darby Canine Kidney) cells, with potential applicability in high-throughput screening (HTS). To this end, we established culture conditions in a collagen matrix, employed forskolin to induce cystogenesis, and tested the effects of rapamycin and birinapant. Forskolin induced cyst formation at different concentrations, and both rapamycin and birinapant reduced cyst number. In conclusion, we successfully developed a miniaturized 3D phenotypic ADPKD model in 384-well plates, with potential applicability HTS-based drug discovery in this disease.
Direction
LOZA GARCIA, MARIA ISABEL (Tutorships)
BREA FLORIANI, JOSE MANUEL (Co-tutorships)
LOZA GARCIA, MARIA ISABEL (Tutorships)
BREA FLORIANI, JOSE MANUEL (Co-tutorships)
Court
ALVAREZ LORENZO, CARMEN ISABEL (Chairman)
YAÑEZ JATO, MATILDE (Secretary)
FONTENLA GIL, JOSE ANGEL (Member)
ALVAREZ LORENZO, CARMEN ISABEL (Chairman)
YAÑEZ JATO, MATILDE (Secretary)
FONTENLA GIL, JOSE ANGEL (Member)
Protamine nanocapsules for the oral co-delivery of curcumin and genetic material as a treatment for inflammatory bowel disease
Authorship
E.A.A.
Master in Drug Research and Development
E.A.A.
Master in Drug Research and Development
Defense date
07.14.2025 10:20
07.14.2025 10:20
Summary
Inflammatory bowel disease, which includes ulcerative colitis and Crohn’s disease, is a chronic condition characterized by exacerbated inflammation mediated by TNF-alfa in the lamina propria. Current treatments based on monoclonal antibodies show low remission rates and require parenteral administration. Therefore, the development of an oral nanosystem combining anti-TNF-alfa siRNA and curcumin is proposed to reduce local inflammation. The vehicles were formulated by solvent displacement and associated with plasmid DNA (pDNA), used as an siRNA model, at 1.5% (w/w). Physicochemical properties, encapsulation efficiency, as well as stability and release in simulated biological media, were evaluated. In vitro studies were also conducted in HeLa and RAW 264.7 cells to assess toxicity, internalization, and transfection efficiency. A monodisperse system with a size below 250 nm, 44% curcumin encapsulation efficiency, and complete plasmid association was obtained. In FaSSIF, the nanosystems demonstrated stability, curcumin release above 20%, and total plasmid release. Finally, their biocompatibility, internalization capacity, and transfection efficiency were confirmed. These results position the vehicles as a promising tool for oral gene therapy in the treatment of inflammatory bowel disease.
Inflammatory bowel disease, which includes ulcerative colitis and Crohn’s disease, is a chronic condition characterized by exacerbated inflammation mediated by TNF-alfa in the lamina propria. Current treatments based on monoclonal antibodies show low remission rates and require parenteral administration. Therefore, the development of an oral nanosystem combining anti-TNF-alfa siRNA and curcumin is proposed to reduce local inflammation. The vehicles were formulated by solvent displacement and associated with plasmid DNA (pDNA), used as an siRNA model, at 1.5% (w/w). Physicochemical properties, encapsulation efficiency, as well as stability and release in simulated biological media, were evaluated. In vitro studies were also conducted in HeLa and RAW 264.7 cells to assess toxicity, internalization, and transfection efficiency. A monodisperse system with a size below 250 nm, 44% curcumin encapsulation efficiency, and complete plasmid association was obtained. In FaSSIF, the nanosystems demonstrated stability, curcumin release above 20%, and total plasmid release. Finally, their biocompatibility, internalization capacity, and transfection efficiency were confirmed. These results position the vehicles as a promising tool for oral gene therapy in the treatment of inflammatory bowel disease.
Direction
CSABA , NOEMI STEFANIA (Tutorships)
CSABA , NOEMI STEFANIA (Tutorships)
Court
ALVAREZ LORENZO, CARMEN ISABEL (Chairman)
YAÑEZ JATO, MATILDE (Secretary)
FONTENLA GIL, JOSE ANGEL (Member)
ALVAREZ LORENZO, CARMEN ISABEL (Chairman)
YAÑEZ JATO, MATILDE (Secretary)
FONTENLA GIL, JOSE ANGEL (Member)
3D Printing by extrusion of Trilostane semi-solids for veterinary use
Authorship
R.A.T.
Master in Drug Research and Development
R.A.T.
Master in Drug Research and Development
Defense date
07.14.2025 13:00
07.14.2025 13:00
Summary
Three-dimensional (3D) printing, developed since the 1980s, allows objects to be manufactured layer by layer from digital models, offering precision in complex designs and reducing costs and time. In the pharmaceutical industry, the FDA has approved 3D-printed drugs, although further studies are still needed to guarantee these processes. Semi-solid extrusion (SSE) is an ideal 3D technique for producing medications, as it uses gel-like textures at low temperatures that maintain the stability of the active ingredient and allow for more precise, customized doses than manual division. This technology is especially valuable in veterinary medicine, where variability in species and weights makes standard dosing difficult, and adaptation of human medications can be unsafe. In this study, customized dosage forms of trilostane for dogs and cats, with doses of 3.5 mg, 14 mg, and 35 mg, were manufactured using semi-solid extrusion 3D printing. Approved excipients and flavorings were added to improve acceptance. The 3D printing system incorporated inline controls that guaranteed uniformity, quality, and absence of defects during manufacturing. The formulations met the European Pharmacopoeia standards for content, weight, and dissolution uniformity, demonstrating precision and reproducibility, with active ingredient release in accordance with regulatory standards. The technology showed great potential for individualized veterinary medicines, with quality controls that optimized small-scale production. 3D printing (SSE) is positioned as an innovative alternative for the development and production of individualized therapies, which requires ongoing research.
Three-dimensional (3D) printing, developed since the 1980s, allows objects to be manufactured layer by layer from digital models, offering precision in complex designs and reducing costs and time. In the pharmaceutical industry, the FDA has approved 3D-printed drugs, although further studies are still needed to guarantee these processes. Semi-solid extrusion (SSE) is an ideal 3D technique for producing medications, as it uses gel-like textures at low temperatures that maintain the stability of the active ingredient and allow for more precise, customized doses than manual division. This technology is especially valuable in veterinary medicine, where variability in species and weights makes standard dosing difficult, and adaptation of human medications can be unsafe. In this study, customized dosage forms of trilostane for dogs and cats, with doses of 3.5 mg, 14 mg, and 35 mg, were manufactured using semi-solid extrusion 3D printing. Approved excipients and flavorings were added to improve acceptance. The 3D printing system incorporated inline controls that guaranteed uniformity, quality, and absence of defects during manufacturing. The formulations met the European Pharmacopoeia standards for content, weight, and dissolution uniformity, demonstrating precision and reproducibility, with active ingredient release in accordance with regulatory standards. The technology showed great potential for individualized veterinary medicines, with quality controls that optimized small-scale production. 3D printing (SSE) is positioned as an innovative alternative for the development and production of individualized therapies, which requires ongoing research.
Direction
GOYANES GOYANES, ALVARO (Tutorships)
GOYANES GOYANES, ALVARO (Tutorships)
Court
LOZA GARCIA, MARIA ISABEL (Chairman)
GARCIA GONZALEZ, CARLOS ALBERTO (Secretary)
CSABA , NOEMI STEFANIA (Member)
LOZA GARCIA, MARIA ISABEL (Chairman)
GARCIA GONZALEZ, CARLOS ALBERTO (Secretary)
CSABA , NOEMI STEFANIA (Member)
Redox-responsive amyloid peptides for the targeted production of antimicrobial nanofibres in bacteria
Authorship
L.A.G.
Master in Drug Research and Development
L.A.G.
Master in Drug Research and Development
Defense date
07.14.2025 10:00
07.14.2025 10:00
Summary
The growing global threat of antibiotic resistance has created an urgent need for new antimicrobial strategies that can overcome traditional resistance mechanisms. In this context, this work aims to develop glycopeptides capable of selectively entering bacterial cells and inducing cytotoxic effects from within. To achieve this, cysteine-containing peptides were synthesized and conjugated to an acetylated glycan moiety through a disulfide bond. The glycan, once deacetylated, improves solubility and facilitates selective internalization, while the disulfide linker allows redox-responsive release of the active peptide in the intracellular environment of bacteria. The experimental work involved the synthesis, purification, and characterisation of different peptide sequences and their glycosylated derivatives. Both solid and solution-phase glycosylation strategies were explored. Deacetylation was performed to restore the native functionality of the glycan. This strategy opens the door to the development of a new class of redox-activated antimicrobial agents, with the potential to bypass conventional resistance pathways.
The growing global threat of antibiotic resistance has created an urgent need for new antimicrobial strategies that can overcome traditional resistance mechanisms. In this context, this work aims to develop glycopeptides capable of selectively entering bacterial cells and inducing cytotoxic effects from within. To achieve this, cysteine-containing peptides were synthesized and conjugated to an acetylated glycan moiety through a disulfide bond. The glycan, once deacetylated, improves solubility and facilitates selective internalization, while the disulfide linker allows redox-responsive release of the active peptide in the intracellular environment of bacteria. The experimental work involved the synthesis, purification, and characterisation of different peptide sequences and their glycosylated derivatives. Both solid and solution-phase glycosylation strategies were explored. Deacetylation was performed to restore the native functionality of the glycan. This strategy opens the door to the development of a new class of redox-activated antimicrobial agents, with the potential to bypass conventional resistance pathways.
Direction
INSUA LOPEZ, IGNACIO (Tutorships)
INSUA LOPEZ, IGNACIO (Tutorships)
Court
LOZA GARCIA, MARIA ISABEL (Chairman)
GARCIA GONZALEZ, CARLOS ALBERTO (Secretary)
CSABA , NOEMI STEFANIA (Member)
LOZA GARCIA, MARIA ISABEL (Chairman)
GARCIA GONZALEZ, CARLOS ALBERTO (Secretary)
CSABA , NOEMI STEFANIA (Member)
Drug nanocarriers with biomimetic coatings
Authorship
S.B.B.
Master in Drug Research and Development
S.B.B.
Master in Drug Research and Development
Defense date
07.14.2025 10:20
07.14.2025 10:20
Summary
Targeted drug delivery remains a key challenge in biomedicine. With the aim of endowing nanoparticles with improved biodistribution capabilities, the process of coating these systems with cell membranes was studied to develop biomimetic nanovehicles. Specifically, the coating of polyethyleneimine (PEI), protamine, and polyphosphazene nanoparticles, cationic systems for which this process is less studied, was investigated. Key parameters such as the number of extrusion cycles and the polymeric core: biological membrane ratio were optimized, resulting in stable, homogeneous systems with low polydispersity. The coating was confirmed through DLS analysis and TEM, SEM, and STEM microscopy. The results demonstrated the feasibility of generating uniformly coated systems with favorable pharmaceutical properties.
Targeted drug delivery remains a key challenge in biomedicine. With the aim of endowing nanoparticles with improved biodistribution capabilities, the process of coating these systems with cell membranes was studied to develop biomimetic nanovehicles. Specifically, the coating of polyethyleneimine (PEI), protamine, and polyphosphazene nanoparticles, cationic systems for which this process is less studied, was investigated. Key parameters such as the number of extrusion cycles and the polymeric core: biological membrane ratio were optimized, resulting in stable, homogeneous systems with low polydispersity. The coating was confirmed through DLS analysis and TEM, SEM, and STEM microscopy. The results demonstrated the feasibility of generating uniformly coated systems with favorable pharmaceutical properties.
Direction
GARCIA FUENTES, MARCOS (Tutorships)
GARCIA FUENTES, MARCOS (Tutorships)
Court
LOZA GARCIA, MARIA ISABEL (Chairman)
GARCIA GONZALEZ, CARLOS ALBERTO (Secretary)
CSABA , NOEMI STEFANIA (Member)
LOZA GARCIA, MARIA ISABEL (Chairman)
GARCIA GONZALEZ, CARLOS ALBERTO (Secretary)
CSABA , NOEMI STEFANIA (Member)
In vitro deconstruction of the indirect impact of gut microbiota alterations on osteoarthritis
Authorship
M.C.S.
Master in Drug Research and Development
M.C.S.
Master in Drug Research and Development
Defense date
07.14.2025 10:40
07.14.2025 10:40
Summary
Osteoarthritis is a degenerative joint disease with a strong inflammatory component, in which intestinal dysbiosis has been identified as a contributing factor. In this context, microbiota alterations can promote the translocation of bacterial lipopolysaccharides (LPS) into the bloodstream, reaching the articular cartilage and activating Toll-like receptor 4 (TLR4), thereby triggering innate immune responses in the joint. In addition to this direct effect, recent studies have linked distant tissues such as hepatic tissue to cartilage inflammation. Since the liver acts as a first-pass organ for gut-derived substances, this study aimed to analyze the effect of different LPS on chondrocytes and hepatocytes, as well as to evaluate the capacity of the secretome from LPS-treated hepatocytes to induce inflammatory responses in chondrocytes. Murine chondrocyte (ATDC5) and human hepatocyte (HepG2) cell lines were treated with LPS from Klebsiella pneumoniae, Acidaminococcus fermentans, Phocaeicola sartorii, Phocaeicola coprophilus, and Escherichia coli (O55:B5 and O26:B6). Inflammatory responses were evaluated by RT-qPCR and colorimetric nitrite assays. Gene expression analysis revealed differential regulation of inflammatory (Il6, Lcn2, Vcam), catabolic (Adamts4, Mmp9), and matrix synthesis (Col2a1) genes, depending on the bacterial species and cell type. Furthermore, the secretome of hepatocytes treated with LPS induced pro-inflammatory responses in chondrocytes. These results support the existence of an indirect mechanism by which LPS promote chondrocyte inflammation via hepatic activation, suggesting a key role for the gut-liver-joint axis in osteoarthritis.
Osteoarthritis is a degenerative joint disease with a strong inflammatory component, in which intestinal dysbiosis has been identified as a contributing factor. In this context, microbiota alterations can promote the translocation of bacterial lipopolysaccharides (LPS) into the bloodstream, reaching the articular cartilage and activating Toll-like receptor 4 (TLR4), thereby triggering innate immune responses in the joint. In addition to this direct effect, recent studies have linked distant tissues such as hepatic tissue to cartilage inflammation. Since the liver acts as a first-pass organ for gut-derived substances, this study aimed to analyze the effect of different LPS on chondrocytes and hepatocytes, as well as to evaluate the capacity of the secretome from LPS-treated hepatocytes to induce inflammatory responses in chondrocytes. Murine chondrocyte (ATDC5) and human hepatocyte (HepG2) cell lines were treated with LPS from Klebsiella pneumoniae, Acidaminococcus fermentans, Phocaeicola sartorii, Phocaeicola coprophilus, and Escherichia coli (O55:B5 and O26:B6). Inflammatory responses were evaluated by RT-qPCR and colorimetric nitrite assays. Gene expression analysis revealed differential regulation of inflammatory (Il6, Lcn2, Vcam), catabolic (Adamts4, Mmp9), and matrix synthesis (Col2a1) genes, depending on the bacterial species and cell type. Furthermore, the secretome of hepatocytes treated with LPS induced pro-inflammatory responses in chondrocytes. These results support the existence of an indirect mechanism by which LPS promote chondrocyte inflammation via hepatic activation, suggesting a key role for the gut-liver-joint axis in osteoarthritis.
Direction
DIAZ RODRIGUEZ, PATRICIA (Tutorships)
Gómez Vaamonde, Rodolfo (Co-tutorships)
DIAZ RODRIGUEZ, PATRICIA (Tutorships)
Gómez Vaamonde, Rodolfo (Co-tutorships)
Court
LOZA GARCIA, MARIA ISABEL (Chairman)
GARCIA GONZALEZ, CARLOS ALBERTO (Secretary)
CSABA , NOEMI STEFANIA (Member)
LOZA GARCIA, MARIA ISABEL (Chairman)
GARCIA GONZALEZ, CARLOS ALBERTO (Secretary)
CSABA , NOEMI STEFANIA (Member)
Topical-ophthalmic formulations of hydroxocobalamin for the treatment of ocular symptoms of homocystinuria type CBLC
Authorship
D.D.L.T.P.
Master in Drug Research and Development
D.D.L.T.P.
Master in Drug Research and Development
Defense date
07.14.2025 11:00
07.14.2025 11:00
Summary
CblC-type homocystinuria is considered a rare genetic disorder that affects cobalamin metabolism and can lead to ocular problems. Hydroxocobalamin (OH-Cbl) is currently the most effective treatment and is administered parenterally, although its delivery to certain ocular regions is limited. The aim of this study was to develop and evaluate ophthalmic formulations based on nanostructured lipid carriers (NLC) loaded with hydroxocobalamin, with the goal of increasing drug availability in the eye. NLCs were prepared using a double emulsion solvent evaporation technique and subsequently characterized in terms of particle size, polydispersity index (PDI), and zeta potential (ζ-potential). Further evaluations included loading capacity, encapsulation efficiency, in vitro release, corneal permeability, drug retention in the cornea, drug distribution between the NLC and the external aqueous phase, and stability in various cell culture media and simulated ophthalmic solutions to determine the overall behavior and stability of the system. The resulting formulations exhibited particle sizes of 279.76 ± 4.66 nm, PDI of 0.241 ± 0.014, ζ-potential of –52.38 ± 1.395 mV, spherical morphology, and high drug encapsulation efficiency (84.1%). They also demonstrated stability under different pH levels, salt concentrations, and biological media. During lyophilization, HP-β-CD was identified as the most suitable cryoprotectant due to its wide use in ophthalmic drugs, high solubility, and excellent ocular tolerability compared to other cryoprotectants. Overall, these results support the potential use of NLCs as a promising alternative for the treatment of ocular symptoms associated with cblC-type homocystinuria
CblC-type homocystinuria is considered a rare genetic disorder that affects cobalamin metabolism and can lead to ocular problems. Hydroxocobalamin (OH-Cbl) is currently the most effective treatment and is administered parenterally, although its delivery to certain ocular regions is limited. The aim of this study was to develop and evaluate ophthalmic formulations based on nanostructured lipid carriers (NLC) loaded with hydroxocobalamin, with the goal of increasing drug availability in the eye. NLCs were prepared using a double emulsion solvent evaporation technique and subsequently characterized in terms of particle size, polydispersity index (PDI), and zeta potential (ζ-potential). Further evaluations included loading capacity, encapsulation efficiency, in vitro release, corneal permeability, drug retention in the cornea, drug distribution between the NLC and the external aqueous phase, and stability in various cell culture media and simulated ophthalmic solutions to determine the overall behavior and stability of the system. The resulting formulations exhibited particle sizes of 279.76 ± 4.66 nm, PDI of 0.241 ± 0.014, ζ-potential of –52.38 ± 1.395 mV, spherical morphology, and high drug encapsulation efficiency (84.1%). They also demonstrated stability under different pH levels, salt concentrations, and biological media. During lyophilization, HP-β-CD was identified as the most suitable cryoprotectant due to its wide use in ophthalmic drugs, high solubility, and excellent ocular tolerability compared to other cryoprotectants. Overall, these results support the potential use of NLCs as a promising alternative for the treatment of ocular symptoms associated with cblC-type homocystinuria
Direction
OTERO ESPINAR, FRANCISCO JAVIER (Tutorships)
DIAZ TOME, VICTORIA (Co-tutorships)
OTERO ESPINAR, FRANCISCO JAVIER (Tutorships)
DIAZ TOME, VICTORIA (Co-tutorships)
Court
LOZA GARCIA, MARIA ISABEL (Chairman)
GARCIA GONZALEZ, CARLOS ALBERTO (Secretary)
CSABA , NOEMI STEFANIA (Member)
LOZA GARCIA, MARIA ISABEL (Chairman)
GARCIA GONZALEZ, CARLOS ALBERTO (Secretary)
CSABA , NOEMI STEFANIA (Member)
Master Dissertation,
Authorship
N.D.M.
Master in Drug Research and Development
N.D.M.
Master in Drug Research and Development
Defense date
07.14.2025 12:00
07.14.2025 12:00
Summary
Advanced cell therapies, such as CAR (Chimeric Antigen Receptor)-T, which redirects T cells against target cells, have revolutionized the treatment of hematological cancers. Another promising strategy is STAb therapy (Secreting T cell-redirecting Antibody), which modifies T cells to locally secrete bispecific antibodies that link immune and tumor cells, promoting their destruction. However, as ex vivo and individualized therapies, their production is complex and costly. To overcome these limitations, this work proposes an innovative strategy: the in vivo modification of T cells using nanoparticles (NPs) encapsulating mRNA encoding for bispecific antibodies, designed to preferentially transfect T cells.This approach would allow the cells to synthesize and secrete the antibodies directly in the organism, replicating the therapeutic effects of STAb through a simpler and more cost-effective procedure. With the aim of directing the NPs towards T cells in vivo,various formulations were designed and characterized. Different lipids and polymeric coatings were tested, and parameters such as size, zeta potential, polydispersity index, encapsulation efficiency, and stability under simulated physiological and storage conditions were evaluated. In addition, toxicity and transfection efficiency were assessed in Jurkat cells and primary T cells. The results confirmed the generation of stable NPs with high encapsulation efficiency, low toxicity, and high transfection efficacy in T cells, laying a solid foundation for future in vivo biodistribution studies and therapeutic evaluations using mRNA coding for bispecific antibodies.
Advanced cell therapies, such as CAR (Chimeric Antigen Receptor)-T, which redirects T cells against target cells, have revolutionized the treatment of hematological cancers. Another promising strategy is STAb therapy (Secreting T cell-redirecting Antibody), which modifies T cells to locally secrete bispecific antibodies that link immune and tumor cells, promoting their destruction. However, as ex vivo and individualized therapies, their production is complex and costly. To overcome these limitations, this work proposes an innovative strategy: the in vivo modification of T cells using nanoparticles (NPs) encapsulating mRNA encoding for bispecific antibodies, designed to preferentially transfect T cells.This approach would allow the cells to synthesize and secrete the antibodies directly in the organism, replicating the therapeutic effects of STAb through a simpler and more cost-effective procedure. With the aim of directing the NPs towards T cells in vivo,various formulations were designed and characterized. Different lipids and polymeric coatings were tested, and parameters such as size, zeta potential, polydispersity index, encapsulation efficiency, and stability under simulated physiological and storage conditions were evaluated. In addition, toxicity and transfection efficiency were assessed in Jurkat cells and primary T cells. The results confirmed the generation of stable NPs with high encapsulation efficiency, low toxicity, and high transfection efficacy in T cells, laying a solid foundation for future in vivo biodistribution studies and therapeutic evaluations using mRNA coding for bispecific antibodies.
Direction
ALONSO FERNANDEZ, MARIA JOSEFA (Tutorships)
Sanjurjo Bouza, Lucía (Co-tutorships)
ALONSO FERNANDEZ, MARIA JOSEFA (Tutorships)
Sanjurjo Bouza, Lucía (Co-tutorships)
Court
LOZA GARCIA, MARIA ISABEL (Chairman)
GARCIA GONZALEZ, CARLOS ALBERTO (Secretary)
CSABA , NOEMI STEFANIA (Member)
LOZA GARCIA, MARIA ISABEL (Chairman)
GARCIA GONZALEZ, CARLOS ALBERTO (Secretary)
CSABA , NOEMI STEFANIA (Member)
Development of an Injectable Hydrogel Containing Nanoparticles for Ocular Administration
Authorship
R.D.G.
Master in Drug Research and Development
R.D.G.
Master in Drug Research and Development
Defense date
07.14.2025 11:00
07.14.2025 11:00
Summary
Age-related Macular Degeneration continues to be one of the leading causes of irreversible blindness in the adult population worldwide. Owing to increased life expectancy, the incidence of this condition is projected to reach nearly 300 million cases by 2040. At present, only the wet form of the disease has available therapeutic options. However, these treatments are solely palliative, aiming to alleviate symptoms and delay disease progression. Furthermore, they require repeated administration due to the rapid release and clearance of the drug within the ocular environment, which can lead to clinical complications, psychological burden for the patient, and substantial economic costs. The application of biomaterials for controlled drug delivery in the treatment of Age-related Macular Degeneration represents a promising strategy. Injectable hydrogels incorporating nanoparticles have emerged as an alternative to the frequent administration of drugs or nanoparticles solutions. In this study, injectable hydrogels composed of chemically modified hyaluronic acid were developed to enable in situ gelation and were loaded with dexamethasone-containing nanocapsules. Morphological and rheological characterizations, degradation assessments and drug release studies were performed. The results indicated that these hydrogels may serve as effective delivery systems to mitigate inflammation associated with the wet form of the disease.
Age-related Macular Degeneration continues to be one of the leading causes of irreversible blindness in the adult population worldwide. Owing to increased life expectancy, the incidence of this condition is projected to reach nearly 300 million cases by 2040. At present, only the wet form of the disease has available therapeutic options. However, these treatments are solely palliative, aiming to alleviate symptoms and delay disease progression. Furthermore, they require repeated administration due to the rapid release and clearance of the drug within the ocular environment, which can lead to clinical complications, psychological burden for the patient, and substantial economic costs. The application of biomaterials for controlled drug delivery in the treatment of Age-related Macular Degeneration represents a promising strategy. Injectable hydrogels incorporating nanoparticles have emerged as an alternative to the frequent administration of drugs or nanoparticles solutions. In this study, injectable hydrogels composed of chemically modified hyaluronic acid were developed to enable in situ gelation and were loaded with dexamethasone-containing nanocapsules. Morphological and rheological characterizations, degradation assessments and drug release studies were performed. The results indicated that these hydrogels may serve as effective delivery systems to mitigate inflammation associated with the wet form of the disease.
Direction
ABBADESSA , ANNA (Tutorships)
ALONSO FERNANDEZ, MARIA JOSEFA (Co-tutorships)
ABBADESSA , ANNA (Tutorships)
ALONSO FERNANDEZ, MARIA JOSEFA (Co-tutorships)
Court
ALVAREZ LORENZO, CARMEN ISABEL (Chairman)
YAÑEZ JATO, MATILDE (Secretary)
FONTENLA GIL, JOSE ANGEL (Member)
ALVAREZ LORENZO, CARMEN ISABEL (Chairman)
YAÑEZ JATO, MATILDE (Secretary)
FONTENLA GIL, JOSE ANGEL (Member)
Preclinical validation of new diagnostic and prognostic biomarkers for Autosomal Dominant Polycystic Kidney Disease
Authorship
L.F.B.
Master in Drug Research and Development
L.F.B.
Master in Drug Research and Development
Defense date
07.14.2025 12:00
07.14.2025 12:00
Summary
Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease, being responsible for 1 in 800-1000 patients worldwide and characterized by the appearance of cysts in the renal parenchyma, which cause end-stage renal failure (ESRD) in the final stages. Despite the existence of multiple diagnostic methods (measurement of creatinine, albumin, genetic studies and imaging techniques), these are not able to detect the disease in its early stages or to indicate its evolution. Therefore, the search for new biomarkers that can fulfill this role is on the rise. One very promising line of research is the use of extracellular vesicles, because they accumulate in their interior multiple metabolites that are indicators of different pathologies. Previous studies in this laboratory have already studied this application, resulting in interest in 4 specific biomarkers found in patient urine samples. This study will focus on the validation of these markers using an animal model (C57/Bl6J Pkd1 flox/flox; TamCre) with 2 inactivation points: early and late, the Western Blot technique and the ExoGAG reagent (Nasasbiotech), which allows the extraction of vesicles from urine samples.
Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease, being responsible for 1 in 800-1000 patients worldwide and characterized by the appearance of cysts in the renal parenchyma, which cause end-stage renal failure (ESRD) in the final stages. Despite the existence of multiple diagnostic methods (measurement of creatinine, albumin, genetic studies and imaging techniques), these are not able to detect the disease in its early stages or to indicate its evolution. Therefore, the search for new biomarkers that can fulfill this role is on the rise. One very promising line of research is the use of extracellular vesicles, because they accumulate in their interior multiple metabolites that are indicators of different pathologies. Previous studies in this laboratory have already studied this application, resulting in interest in 4 specific biomarkers found in patient urine samples. This study will focus on the validation of these markers using an animal model (C57/Bl6J Pkd1 flox/flox; TamCre) with 2 inactivation points: early and late, the Western Blot technique and the ExoGAG reagent (Nasasbiotech), which allows the extraction of vesicles from urine samples.
Direction
Sánchez Barreiro, Alejandro (Tutorships)
García González, Miguel Ángel (Co-tutorships)
Sánchez Barreiro, Alejandro (Tutorships)
García González, Miguel Ángel (Co-tutorships)
Court
ALVAREZ LORENZO, CARMEN ISABEL (Chairman)
YAÑEZ JATO, MATILDE (Secretary)
FONTENLA GIL, JOSE ANGEL (Member)
ALVAREZ LORENZO, CARMEN ISABEL (Chairman)
YAÑEZ JATO, MATILDE (Secretary)
FONTENLA GIL, JOSE ANGEL (Member)
Development of a Tumor-on-a-Chip to evaluate dendritic cell migration to breast tumors.
Authorship
S.D.P.G.R.
Master in Drug Research and Development
S.D.P.G.R.
Master in Drug Research and Development
Defense date
07.14.2025 12:20
07.14.2025 12:20
Summary
The tumor microenvironment plays a fundamental role in cancer progression, immune evasion and treatment resistance. Accurately replicating this complex environment remains a major challenge for the development of new therapeutic strategies. In this context, three-dimensional hydrogel-based models offer a promising alternative to traditional 2D cultures and animal models, as they better mimic the physiological conditions of real tumors. This work focuses on developing a 3D model as a starting point for a Tumor-on-a-Chip system, using type I collagen hydrogels as the matrix. The system integrates breast cancer cells (MDA-MB-231), macrophages derived from the THP-1 cell line, and dendritic cells obtained from human blood. This combination aims to recreate an immunologically relevant tumor microenvironment that enables the study of key processes, such as dendritic cell migration toward the breast tumor. The model is designed to evaluate viability, proliferation, morphology and functional behaviour of the different cell populations, as well as their interactions within the matrix. In addition, the model also considers the possibility of tuning collagen matrix degradation, enabling dynamic control of the tumor microenvironment. This strategy offers an innovative tool to investigate the tumor microenvironment in breast cancer.
The tumor microenvironment plays a fundamental role in cancer progression, immune evasion and treatment resistance. Accurately replicating this complex environment remains a major challenge for the development of new therapeutic strategies. In this context, three-dimensional hydrogel-based models offer a promising alternative to traditional 2D cultures and animal models, as they better mimic the physiological conditions of real tumors. This work focuses on developing a 3D model as a starting point for a Tumor-on-a-Chip system, using type I collagen hydrogels as the matrix. The system integrates breast cancer cells (MDA-MB-231), macrophages derived from the THP-1 cell line, and dendritic cells obtained from human blood. This combination aims to recreate an immunologically relevant tumor microenvironment that enables the study of key processes, such as dendritic cell migration toward the breast tumor. The model is designed to evaluate viability, proliferation, morphology and functional behaviour of the different cell populations, as well as their interactions within the matrix. In addition, the model also considers the possibility of tuning collagen matrix degradation, enabling dynamic control of the tumor microenvironment. This strategy offers an innovative tool to investigate the tumor microenvironment in breast cancer.
Direction
ALVAREZ LORENZO, CARMEN ISABEL (Tutorships)
BLANCO FERNANDEZ, BARBARA (Co-tutorships)
ALVAREZ LORENZO, CARMEN ISABEL (Tutorships)
BLANCO FERNANDEZ, BARBARA (Co-tutorships)
Court
LOZA GARCIA, MARIA ISABEL (Chairman)
GARCIA GONZALEZ, CARLOS ALBERTO (Secretary)
CSABA , NOEMI STEFANIA (Member)
LOZA GARCIA, MARIA ISABEL (Chairman)
GARCIA GONZALEZ, CARLOS ALBERTO (Secretary)
CSABA , NOEMI STEFANIA (Member)
Identification of compounds protective against neurite loss in a phenotypic model of familial Alzheimer’s.
Authorship
E.L.R.
Master in Drug Research and Development
E.L.R.
Master in Drug Research and Development
Defense date
07.14.2025 12:20
07.14.2025 12:20
Summary
Familial Alzheimer’s disease (AD), caused by mutations such as APP V717I and MAPT P301L, is characterized, among other features, by the loss of neuritic connections. In this study, a phenotypic cellular model of AD was optimized using differentiated SH-SY5Y cells transfected with both mutations, with the aim of assessing neurite loss and its possible pharmacological reversal. Transfection significantly reduced the percentage of cells with neurites, reproducing morphological alterations compatible with neurodegenerative processes. Using this model, seven repurposed drugs were tested, selected for their neuroprotective effects in previous excitability assays. Among them, melatonin showed a protective effect, increasing the number of cells with neurites compared to the control. In contrast, metolazone and rifampicin further reduced the number of neurite-bearing cells. Mianserin, ethosuximide, doxepin, and canavanine did not significantly affect the number of arborized cells under the evaluated conditions. This study demonstrates that the model is suitable for detecting the loss of neurite-bearing cells and for identifying compounds with potential neuroprotective effects against such loss. The results will need to be validated in more complex models to confirm their pathophysiological relevance.
Familial Alzheimer’s disease (AD), caused by mutations such as APP V717I and MAPT P301L, is characterized, among other features, by the loss of neuritic connections. In this study, a phenotypic cellular model of AD was optimized using differentiated SH-SY5Y cells transfected with both mutations, with the aim of assessing neurite loss and its possible pharmacological reversal. Transfection significantly reduced the percentage of cells with neurites, reproducing morphological alterations compatible with neurodegenerative processes. Using this model, seven repurposed drugs were tested, selected for their neuroprotective effects in previous excitability assays. Among them, melatonin showed a protective effect, increasing the number of cells with neurites compared to the control. In contrast, metolazone and rifampicin further reduced the number of neurite-bearing cells. Mianserin, ethosuximide, doxepin, and canavanine did not significantly affect the number of arborized cells under the evaluated conditions. This study demonstrates that the model is suitable for detecting the loss of neurite-bearing cells and for identifying compounds with potential neuroprotective effects against such loss. The results will need to be validated in more complex models to confirm their pathophysiological relevance.
Direction
LOZA GARCIA, MARIA ISABEL (Tutorships)
MARTINEZ RODRIGUEZ, ANTON LEANDRO (Co-tutorships)
LOZA GARCIA, MARIA ISABEL (Tutorships)
MARTINEZ RODRIGUEZ, ANTON LEANDRO (Co-tutorships)
Court
ALVAREZ LORENZO, CARMEN ISABEL (Chairman)
YAÑEZ JATO, MATILDE (Secretary)
FONTENLA GIL, JOSE ANGEL (Member)
ALVAREZ LORENZO, CARMEN ISABEL (Chairman)
YAÑEZ JATO, MATILDE (Secretary)
FONTENLA GIL, JOSE ANGEL (Member)
Activation profile of the JAK/STAT pathway in patients with ulcerative colitis: interindividual variability and sex-related differences.
Authorship
A.R.L.
Master in Drug Research and Development
A.R.L.
Master in Drug Research and Development
Defense date
07.14.2025 12:40
07.14.2025 12:40
Summary
Ulcerative colitis is characterized by chronic inflammation of the colonic mucosa in which the JAK/STAT pathway plays a central role. In this study, we quantified the phosphorylation of seven key mediators of this pathway (JAK1, JAK2, JAK3, TYK2, STAT1, STAT3, and STAT4) in inflamed colonic biopsies from seven young UC patients with homogeneous clinical features (MES = 2, mesalazine monotherapy). These patients were selected for preliminary analyses intended to inform hypothesis generation. Marked inter-individual variability emerged, although JAK2 was the component that showed the highest activation level, followed by TYK2 and STAT4. Moreover, correlation analyses revealed significant associations between TYK2 and JAK3, TYK2 and STAT3/4, and JAK2 and STAT4, indicating coordinated activation of these signaling nodes. Phosphorylation levels of all mediators, except STAT1, were significantly higher in males, suggesting that it may exist a sex-dependent modulation of JAK/STAT signaling. These findings underscore the value of individual molecular profiling to explain clinical heterogeneity in UC and to look forward biomarkers to guide more personalized therapeutic strategies.
Ulcerative colitis is characterized by chronic inflammation of the colonic mucosa in which the JAK/STAT pathway plays a central role. In this study, we quantified the phosphorylation of seven key mediators of this pathway (JAK1, JAK2, JAK3, TYK2, STAT1, STAT3, and STAT4) in inflamed colonic biopsies from seven young UC patients with homogeneous clinical features (MES = 2, mesalazine monotherapy). These patients were selected for preliminary analyses intended to inform hypothesis generation. Marked inter-individual variability emerged, although JAK2 was the component that showed the highest activation level, followed by TYK2 and STAT4. Moreover, correlation analyses revealed significant associations between TYK2 and JAK3, TYK2 and STAT3/4, and JAK2 and STAT4, indicating coordinated activation of these signaling nodes. Phosphorylation levels of all mediators, except STAT1, were significantly higher in males, suggesting that it may exist a sex-dependent modulation of JAK/STAT signaling. These findings underscore the value of individual molecular profiling to explain clinical heterogeneity in UC and to look forward biomarkers to guide more personalized therapeutic strategies.
Direction
LOZA GARCIA, MARIA ISABEL (Tutorships)
MARTINEZ RODRIGUEZ, ANTON LEANDRO (Co-tutorships)
LOZA GARCIA, MARIA ISABEL (Tutorships)
MARTINEZ RODRIGUEZ, ANTON LEANDRO (Co-tutorships)
Court
ALVAREZ LORENZO, CARMEN ISABEL (Chairman)
YAÑEZ JATO, MATILDE (Secretary)
FONTENLA GIL, JOSE ANGEL (Member)
ALVAREZ LORENZO, CARMEN ISABEL (Chairman)
YAÑEZ JATO, MATILDE (Secretary)
FONTENLA GIL, JOSE ANGEL (Member)
Study of the alterations in the blood-brain barrier produced by the secretory phenotype associated with senescence of peripheral vascular endothelial cells
Authorship
L.R.R.
Master in Drug Research and Development
L.R.R.
Master in Drug Research and Development
Defense date
07.14.2025 12:40
07.14.2025 12:40
Summary
Age is the main risk factor for the development of neurodegenerative diseases. A common feature among these pathologies is the disruption of the blood-brain barrier (BBB), a key factor in their onset and/or progression. Despite advances in our understanding of the BBB role, few studies have explored the relationship between peripheral vascular alterations and BBB disruption. Peripheral vasculature also undergoes age-related changes, such as cellular senescence, a state in which cells acquire a pro-inflammatory phenotype known as the senescence-associated secretory phenotype (SASP), which can affect distant functions, including BBB integrity. The aim of this study was to evaluate the impact of endothelial SASP on an in vitro BBB model based on the bEnd.3 cell line, as well as to assess the potential protective effect of rapamycin. For this purpose, cells were exposed to different conditioned media obtained from senescent HUVECs at various passages, either treated or untreated with rapamycin. The following parameters were analysed: cell viability, oxidative stress, levels of pro-inflammatory mediators (IL-6), expression of phosphorylated endothelial nitric oxide synthase (p-eNOS), transendothelial electrical resistance (TEER), and the expression of tight junction proteins (TJs) (claudin-5 and occludin). The results showed that conditioned medium from rapamycin-treated cells reduced oxidative stress and IL-6 levels, increased p-eNOS expression, and improved both TEER and the expression of TJ proteins. These findings highlight the potential of rapamycin as a therapy to reduce the effects of vascular aging on neurovascular dysfunction, a central link between cardiovascular disease and neurodegeneration.
Age is the main risk factor for the development of neurodegenerative diseases. A common feature among these pathologies is the disruption of the blood-brain barrier (BBB), a key factor in their onset and/or progression. Despite advances in our understanding of the BBB role, few studies have explored the relationship between peripheral vascular alterations and BBB disruption. Peripheral vasculature also undergoes age-related changes, such as cellular senescence, a state in which cells acquire a pro-inflammatory phenotype known as the senescence-associated secretory phenotype (SASP), which can affect distant functions, including BBB integrity. The aim of this study was to evaluate the impact of endothelial SASP on an in vitro BBB model based on the bEnd.3 cell line, as well as to assess the potential protective effect of rapamycin. For this purpose, cells were exposed to different conditioned media obtained from senescent HUVECs at various passages, either treated or untreated with rapamycin. The following parameters were analysed: cell viability, oxidative stress, levels of pro-inflammatory mediators (IL-6), expression of phosphorylated endothelial nitric oxide synthase (p-eNOS), transendothelial electrical resistance (TEER), and the expression of tight junction proteins (TJs) (claudin-5 and occludin). The results showed that conditioned medium from rapamycin-treated cells reduced oxidative stress and IL-6 levels, increased p-eNOS expression, and improved both TEER and the expression of TJ proteins. These findings highlight the potential of rapamycin as a therapy to reduce the effects of vascular aging on neurovascular dysfunction, a central link between cardiovascular disease and neurodegeneration.
Direction
CAMPOS TOIMIL, MANUEL (Tutorships)
VIÑA CASTELAO, MARÍA DOLORES (Co-tutorships)
CAMPOS TOIMIL, MANUEL (Tutorships)
VIÑA CASTELAO, MARÍA DOLORES (Co-tutorships)
Court
LOZA GARCIA, MARIA ISABEL (Chairman)
GARCIA GONZALEZ, CARLOS ALBERTO (Secretary)
CSABA , NOEMI STEFANIA (Member)
LOZA GARCIA, MARIA ISABEL (Chairman)
GARCIA GONZALEZ, CARLOS ALBERTO (Secretary)
CSABA , NOEMI STEFANIA (Member)
Pharmacological control of innate immunity linked to the gut microbiota in cellular models of rheumatoid arthritis
Authorship
L.Z.V.
Master in Drug Research and Development
L.Z.V.
Master in Drug Research and Development
Defense date
07.14.2025 13:00
07.14.2025 13:00
Summary
Rheumatoid arthritis (RA) is one of the most common autoimmune diseases and is primarily characterized by chronic inflammation of the synovial membrane, triggered by an abnormal immune response against the body's own joint tissues. It has been suggested that alterations in the intestinal microbiota may contribute to the development of endotoxemia through the translocation of bacterial lipopolysaccharides (LPS) into the bloodstream, a phenomenon that has been linked to RA. In this context, these LPS could reach the joints, worsening inflammation by activating Toll-like receptor 4 (TLR4), a key component of the innate immune system. This study analyzes TLR4-mediated inflammatory responses in fibroblast-like synoviocytes and macrophages after the exposure to LPS from different intestinal bacteria and evaluates the potential anti-inflammatory effect of the repurposed drugs amitriptyline and FX in an in vitro model of RA. Cell lines were treated with LPS from Escherichia coli, Klebsiella pneumoniae, Acidaminococcus fermentans, Phocaeicola sartorii, and Phocaeicola coprophilus. The inflammatory response was assessed by RT-qPCR and protein analysis via capillary electrophoresis. The results indicate that the inflammatory response varies depending on the cell type and the bacterial species used as stimulus. In macrophages, A.fermentans induced the highest inflammatory response, while in synoviocytes, the most pronounced effects were observed with P.sartorii and P.coprophilus. FX showed anti-inflammatory efficacy exclusively in macrophages, whereas amitriptyline had no effect in either cell type. These findings highlight the importance of developing new strategies to modulate the immune response in RA and exploring therapeutic combinations that enhance anti-inflammatory effects.
Rheumatoid arthritis (RA) is one of the most common autoimmune diseases and is primarily characterized by chronic inflammation of the synovial membrane, triggered by an abnormal immune response against the body's own joint tissues. It has been suggested that alterations in the intestinal microbiota may contribute to the development of endotoxemia through the translocation of bacterial lipopolysaccharides (LPS) into the bloodstream, a phenomenon that has been linked to RA. In this context, these LPS could reach the joints, worsening inflammation by activating Toll-like receptor 4 (TLR4), a key component of the innate immune system. This study analyzes TLR4-mediated inflammatory responses in fibroblast-like synoviocytes and macrophages after the exposure to LPS from different intestinal bacteria and evaluates the potential anti-inflammatory effect of the repurposed drugs amitriptyline and FX in an in vitro model of RA. Cell lines were treated with LPS from Escherichia coli, Klebsiella pneumoniae, Acidaminococcus fermentans, Phocaeicola sartorii, and Phocaeicola coprophilus. The inflammatory response was assessed by RT-qPCR and protein analysis via capillary electrophoresis. The results indicate that the inflammatory response varies depending on the cell type and the bacterial species used as stimulus. In macrophages, A.fermentans induced the highest inflammatory response, while in synoviocytes, the most pronounced effects were observed with P.sartorii and P.coprophilus. FX showed anti-inflammatory efficacy exclusively in macrophages, whereas amitriptyline had no effect in either cell type. These findings highlight the importance of developing new strategies to modulate the immune response in RA and exploring therapeutic combinations that enhance anti-inflammatory effects.
Direction
DIAZ RODRIGUEZ, PATRICIA (Tutorships)
Gómez Vaamonde, Rodolfo (Co-tutorships)
DIAZ RODRIGUEZ, PATRICIA (Tutorships)
Gómez Vaamonde, Rodolfo (Co-tutorships)
Court
ALVAREZ LORENZO, CARMEN ISABEL (Chairman)
YAÑEZ JATO, MATILDE (Secretary)
FONTENLA GIL, JOSE ANGEL (Member)
ALVAREZ LORENZO, CARMEN ISABEL (Chairman)
YAÑEZ JATO, MATILDE (Secretary)
FONTENLA GIL, JOSE ANGEL (Member)