Tesis Doctorals - Facultat - Biologia

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Intrinsic tracers for the analysis of habitat use by large marine vertebrates
(Universitat de Barcelona, 2025-04-01) Cani, Alessandra; Cardona Pascual, Luis; Drago, Massimiliano; Universitat de Barcelona. Facultat de Biologia
[eng] The use of the stable isotopes of different chemical elements as intrinsic biogeochemical tracers offers a unique opportunity to infer information about the foraging and spatial ecology of large marine vertebrates from small tissue samples of alive or dead individuals. Historically, the most commonly used tracers to study the trophic ecology of marine species have been the stable isotope ratios of carbon (δ13C) and nitrogen (δ15N), since they allow to identify the contribution of different primary producers with distinct isotopic signatures to the diet of a consumer and to estimate the trophic position of species and individuals within an ecosystem. More recently, the use of these isotopic tracers to analyse the habitat use of marine animals has led to the inclusion of other chemical elements to explore new dimensions of the isotopic niche. Such is the case of the sulphur stable isotope ratio (δ34S) to differentiate between inshore/benthic habits from offshore/pelagic habits, and the oxygen stable isotope ratio (δ18O) to study migration patterns across areas with distinct salinity. Nevertheless, the advantages of using additional isotopic tracers to improve the definition of the isotopic niche will depend on the ecosystem and species involved and, particularly, on the existence and scale of environmental isotopic gradients that affect the stable isotope ratios in primary producers and along the respective food webs. Therefore, this thesis aims to assess the suitability of the δ34S and δ18O ratio as habitat tracers for two different aspects, (1) to evaluate the isotopic niche partitioning among species of the same community and (2) to identify individual movement patterns among isotopically distinct foraging grounds. To do this, the δ13C, δ15N, δ34S and/or δ18O ratios were analysed in two types of consumer tissues, bone of marine mammals and sea turtles and epidermis of sea turtles, inhabiting ecosystems with marked environmental isotopic gradients such as the Río de la Plata estuary, the Mauritanian coast and the North Atlantic Ocean-Mediterranean Sea system. On one hand, the addition of the δ18O and δ34S ratios to the δ13C and δ15N ratios allowed to better characterize the isotopic niche partitioning among marine consumers in an ecosystem with important inputs of freshwater and terrestrial particulate organic matter, since the spatial scale of the environmental isotopic gradients covered the area used by the species. However, this did not occur everywhere and hence, the inclusion of more tracers does not necessarily translate into an improvement of the characterization of the isotopic niche partitioning among species. On the other hand, the use of at least three of these intrinsic tracers allowed to identify individual specialization in habitat within populations, as well as to trace individual movements among isotopically distinct areas. Furthermore, accounting for as many physical and chemical processes as possible, both environmental and metabolic, is critical when using stable isotopes to assess the habitat use and trophic ecology of large marine vertebrates, as the isotopic ratios of the consumers can be jointly affected by many processes. Finally, results should be validated with other complementary research techniques, such as direct observations, satellite telemetry and genetic analysis.
Decoding Functional Genomics: Exploring Gene Expression Regulation and Unveiling the Drivers of Inter-Individual Variation
(Universitat de Barcelona, 2025-04-07) Oliveros Diez, Winona; Melé Messeguer, Marina; Universitat de Barcelona. Facultat de Biologia
[eng] Understanding gene expression and its regulation is crucial for uncovering the molecular mechanisms driving phenotypic diversity and disease susceptibility. Regulatory regions of the genome, including non-coding elements, play a pivotal role in controlling gene activity, yet their influence on transcriptomic variation remains incompletely understood. My work, conducted within the Transcriptomics and Functional Genomics group at the Barcelona Supercomputing Center under the guidance of Dr. Marta Melé, aims to unravel these complexities by leveraging high-throughput sequencing technologies and advanced computational analyses. This PhD thesis explores the regulatory mechanisms of the human transcriptome in health and disease, employing both large-scale transcriptomic analyses and functional genomics approaches. It consists of two main chapters, each addressing distinct aspects of gene regulation using complementary methodologies. The main goal is to elucidate how genetic, demographic, and environmental factors shape transcriptional and epigenetic regulation, ultimately contributing to phenotypic diversity and disease susceptibility. These insights pave the way for more effective and personalized medical approaches. The thesis is structured into two main chapters. In Chapter 1, we investigate how demographic factors (such as sex, age, ancestry, and BMI) collectively influence gene expression, alternative splicing, and DNA methylation across multiple human tissues. While previous transcriptomic studies have provided valuable insights into gene regulation, the full extent of inter-individual variability remains poorly characterized. Using large-scale publicly available datasets, we systematically quantify the contributions of these demographic traits, identify common and tissue-specific regulatory patterns, and explore the potential functional consequences of DNA methylation variations. Our findings reveal widespread hypermethylation in the female autosomal genome, particularly at Polycomb-repressed regions, across tissues. Additionally, we observe a systematic age-related hypermethylation pattern at Polycomb target regions and regulatory sites associated with developmental genes in both sexes across tissues, with the exception of the gonads. Furthermore, ancestry-related differences, particularly in ribosomal protein regulation and the epigenetic landscape, underscore the importance of population diversity in genomic studies. These insights highlight the need for inclusive research frameworks to better understand human biology and disease mechanisms across populations. In Chapter 2, we shift focus to the functional effects of non-coding genetic variants associated with cardiovascular traits. Many disease-associated variants identified through genome-wide association studies (GWAS) lie within regulatory regions, yet their precise roles remain unclear. To address this, we employ Massively Parallel Reporter Assays (MPRAs) to functionally assess 4,608 genetic variants linked to blood pressure regulation in vascular smooth muscle cells (VSMCs) and cardiomyocytes (CMs). By integrating MPRA results with publicly available epigenomic datasets, we refine a set of likely regulatory variants and identify their potential roles in cardiovascular disease. Our findings highlight the complexity of non-coding variant function, demonstrating that regulatory effects are often cell type-specific. This work not only advances our understanding of the genetic architecture of cardiovascular disease but also lays the foundation for future research aimed at developing targeted therapies. By combining large-scale transcriptomic analyses with high-throughput functional assays, this thesis provides a comprehensive view of gene regulation in both health and disease contexts. This research underscores the importance of considering demographic diversity in transcriptomic studies and highlights the power of functional genomics approaches in deciphering the roles of non-coding variants. These findings have important implications for personalized medicine, contributing to a deeper understanding of the molecular mechanisms underlying disease susceptibility.
In utero green space exposure, DNA methylation, and birth size metrics
(2025-03-24) Aguilar Lacasaña, Sofía; Vrijheid, Martine; Bustamante Pineda, Mariona; Universitat de Barcelona. Facultat de Biologia
[eng] An increasing body of evidence has established health benefits of exposure to green space. Notably, in utero exposure to green space has been linked to a reduced risk of adverse birth outcomes, such as low birth weight (LBW), a critical indicator of long-term heath throughout the life-course. Despite these associations, the biological mechanisms underlying the benefits of green space remain unclear, with epigenetic changes emerging as a potential explanation. The aim of this thesis was to investigate the epigenetic mechanisms, focusing on DNA methylation (DNAm) patterns, associated with green space exposure and birth size metrics. First, we examined the association between green space exposure and DNAm through epigenome-wide association studies (EWAS), focused on two tissue types: blood and placenta. In blood, we analyzed two exposure windows using data from eight European birth cohorts with a total of 2,988 newborns and 1,849 children. After multiple-testing correction, in utero greenness exposure was associated with three differentially methylated regions (DMRs) in cord blood. These regions encompass the SLC6A12, ADAMTS2, and KCNQ1DN genes, with the latter two previously reported in the literature in relation to green space exposure. These genes regulate extracellular matrix organization, blood vessel development, cell growth inhibition, and TGF-beta receptor signaling. The cumulative exposure to greenness from conception to childhood was associated with one DMR in the SDK1 gene, related to immune regulation and neural development. None of the exposure windows was associated with individual differentially methylated positions (DMPs). In the placenta, data from 550 mother-child pairs from the Barcelona Life Study Cohort (BiSC) revealed a significant association between residential greenness and DNAm levels at a DMP located in the SLC25A10 gene. This gene is linked to metabolic processes and body mass index (BMI). Additional suggestive DMPs were identified, related to pathways involving glucocorticoid signaling, inflammatory responses, and oxidative stress. No DMRs were detected after adjusting for multiple testing and additional checks. Second, we explored the association between placental DNAm and birth size metrics, including birth weight (BW), birth length (BL), birth weight-for-length (WLR), and head circumference (HC) in 3,852 neonates from 16 international cohorts. DNAm at 1,106 CpGs was associated with at least one of the birth size metrics, with 1,000 of them being associated with BW. Through positional annotation, promoter-enhancer interactions, and expression quantitative trait methylations (eQTMs), key pathways for placental function and fetal growth were identified, including glucose metabolism, growth and hormonal signaling, immune regulation, vascularization, and hypoxia, as well as enrichment for certain placental transcription factors (TFs). Moreover, sex specific differences in DNAm patterns related to BW were observed, with nine female specific and 38 male-specific DMPs. Additionally, the comparison of the findings with those previously reported for cord blood indicated tissue-specific effects. Furthermore, we identified seven cell-dependent DMPs. The genetic contribution was also assessed, revealing that approximately 20% of the DMPs were influenced by fetal methylation quantitative trait loci acting in cis (cis-mQTLs). To infer causality between the DNAm and BW, bi-directional Mendelian randomization (MR) analyses were conducted, suggesting that DNAm at nine DMPs might affect BW, though further studies are needed to exclude horizontal pleiotropy. Potential causal CpGs were annotated to genes associated with placental development and function as well as obstetric complications (RASSF1, SEMA3F, SIK3, SLC2A4RG). Finally, we investigated whether placental DNAm markers could underlie the association between in utero exposure to green space and BW by applying the meet-in-middle approach. We observed that 37 out of the 1,000 BW-associated DMPs had consistent associations with green space exposure, suggesting a potential mediating role. Experimental or interventional studies are necessary to confirm causality. In summary, this thesis, through international multi-cohort studies, has elucidated placental epigenetic mechanisms involved in fetal growth. It also has pointed to biological pathways that aligned with benefits of green space, such as mental restoration and physical activity. Results, however, should be interpreted cautiously, and future research with more precise exposure assessments, cellular models, and interventional designs will be essential to fully confirm these findings.
From stem cells to cancer: novel methods for tracking clonal behavior and tumour heterogeneity
(Universitat de Barcelona, 2025-03-24) Singh, Indranil; Rodriguez-Fraticelli, Alejo E.; Universitat de Barcelona. Facultat de Biologia
[eng] Hematopoietic stem and progenitor cells serve as a cornerstone of tissue homeostasis, enabling the generation of diverse blood cell types and immune responses. However, aberrations in their clonal dynamics and differentiation trajectories underpin a range of pathological conditions, including hematological malignancies, cardiovascular diseases, and tumor progression. This thesis employs advanced single-cell and lineage-tracing methodologies to dissect the cellular and molecular mechanisms governing stem cell behavior, differentiation, and functional diversity across disease contexts. In the first study, we elucidate the role of the cellular state of origin in dictating leukemic phenotypes. Using clonal tracing in mouse models, we demonstrate that low fitness stem cells, typically outcompeted during normal hematopoiesis, gain a competitive advantage following mutations in Dnmt3a-R878H or Npm1c. These mutations not only amplify specific clones but also reprogram their differentiation trajectories, underscoring the role of clonal "reaction norms" in cancer heterogeneity. The second study presents EPI-clone, a transgene-free lineage-tracing framework leveraging somatic epimutations to resolve clonal dynamics at single-cell resolution. Applied to murine and human hematopoiesis, EPI-clone reveals that age-related myeloid bias arises from a restricted pool of expanded clones, while many functionally young-like clones persist. In humans, we identify a spectrum of age-related clonal expansions, both with and without driver mutations, establishing a continuum of clonal hematopoiesis in ageing. The third study investigates the role of hematopoietic stem cells (HSCs) in emergency hematopoiesis (EH) following myocardial infarction (MI). By profiling human bone marrow from cardiac surgery patients, we uncover transcriptional and functional shifts in HSCs that promote pro-inflammatory myelopoiesis, contributing to impaired cardiac repair. Therapeutically, enforcing HSC quiescence via 4-oxo-retinoic acid mitigates inflammatory myelopoiesis, preserving cardiac function post-MI and providing a potential strategy for modulating excessive immune responses. Finally, the fourth study explores neutrophil development and its implications for cancer progression. We identify the cationic amino acid transporter Slc7a7 as a critical regulator of neutrophil maturation and function. Loss of Slc7a7 disrupts differentiation trajectories, reduces pro-inflammatory responses, and diminishes neutrophil extracellular trap (NET) formation. Remarkably, Slc7a7 deficiency delays tumor progression in a melanoma model, highlighting amino acid metabolism as a promising therapeutic target to modulate neutrophil responses in inflammatory diseases and cancer. Collectively, this thesis provides a comprehensive exploration of stem and progenitor cell biology across diverse pathological contexts, offering novel insights into clonal dynamics, differentiation plasticity, and therapeutic interventions. These findings advance our understanding of the cellular hierarchies and molecular pathways that underpin disease progression, with implications for targeted therapies in cancer, cardiovascular disease, and immune dysregulation.
Dissecting TGFβ-Driven Resistance to αPD-L1 Therapy in Colorectal Cancer Liver Metastases. Enhanced T Cell Motility and Proliferation for Effective Anti-Tumor Responses
(Universitat de Barcelona, 2025-01-16) Salvany Celades, Maria; Batlle Gómez, Eduard; Prados Martín, Alejandro; Universitat de Barcelona. Facultat de Biologia
[eng] Colorectal cancer (CRC), particularly its metastatic forms, poses signiﬁcant therapeutic challenges, with immune checkpoint blockade (ICB) therapies providing limited beneﬁts to patients whose metastasis lack T cell inﬁltration. This highlights the need to target mechanisms underlying T cell exclusion to improve clinical outcomes. TGF-β signaling plays a pivotal role in regulating the tumor microenvironment (TME) and promoting immune suppression. Using experimental models of metastatic CRC, we demonstrate how TGF-β inhibition enables immune cell inﬁltration and enhances the efﬁcacy of ICB therapy. Our ﬁndings reveal that TGF-β imposes dual immunosuppressive barriers by preventing the migration of memory-like CD8+ T cells from the periphery into the TME and by directing macrophages to suppress the clonal expansion T cells within metastases. We also observed that while TGF-β signaling drives the formation of a dense, collagen-rich extracellular matrix (ECM), it does not inherently restrict T cell motility within the tumor. Instead, PD-1/PD-L1 interactions primarily regulate T cell movement in the TME. Moreover, our study identiﬁes a key role for TGF-β in instructing tumor-associated macrophages (TAMs) to adopt an immunosuppressive phenotype characterized by SPP1 expression, contributing to immune evasion and metastasis. The TGF-β–SPP1 axis was found to correlate with poor prognosis in CRC patients, emphasizing the therapeutic potential of targeting SPP1+ macrophages to overcome immune suppression. Overall, this study provides novel insights into the mechanisms of TGF-β -mediated immune evasion and highlights the importance of targeting TGF-β signaling in both peripheral T cells and TAMs to improve the efﬁcacy of ICB therapies in metastatic CRC.
Understanding, predicting and preventing the impact of nonsense mutations on gene expression by deep mutational scanning
(Universitat de Barcelona, 2025-01-17) Toledano Martin, Ignasi; Supek, Fran; Lehner, Ben, 1978-; Universitat de Barcelona. Facultat de Biologia
[eng] Premature termination codons (PTCs) are responsible for ~10–20% of inherited diseases and represent a major mechanism of tumor suppressor gene inactivation in cancer. Traditionally, PTCs are considered to induce transcript degradation via nonsense-mediated mRNA decay (NMD) and lead to the production of truncated non-functional proteins. Nonsense suppression therapies aim to promote translational readthrough over PTCs, enabling the synthesis of full-length proteins. Both NMD and readthrough modulate the severity of disease phenotypes by regulating the abundance of the mRNA and the full-length protein; respectively. However, their efficiencies vary across PTCs. In this thesis, we employed deep mutagenesis methods to systematically quantify how sequence context and other genetic factors influence the mRNA levels and the full-length protein abundance of PTC-containing transcripts. First, we developed a methodological improvement for deep mutagenesis libraries generation. Second, a comprehensive assessment of drug-induced readthrough was performed, encompassing over 140,000 measurements and generating readthrough scores for 6,000 PTCs that cause genetic diseases and cancer. This massive dataset was subsequently leveraged to elucidate the effect of sequence context on readthrough and to train accurate predictive models to estimate drug-specific PTC readthrough genome-wide. We envisage these datasets will become a valuable resource to improve clinical trial design and the development of personalized nonsense suppression therapies. Third, we combined different libraries to test and extend hypotheses for how PTC position, exon length, sequence context and translation reinitiation interplay to determine NMD efficiency. Overall, this thesis provides a comprehensive view of how the sequence landscape influences the fate of PTC-containing transcripts. More broadly, it demonstrates the effectiveness of deep mutagenesis in uncovering sequence-to- activity relationships, highlighting the potential of this approach for investigating other mRNA-related processes.
CPEB1 and CPEB4 in skin homeostasis and tumorigenesis
(Universitat de Barcelona, 2025-01-24) Ferrer Caelles, Anna; Méndez de la Iglesia, Raúl; Universitat de Barcelona. Facultat de Biologia
[eng] The skin epidermis is a continuously renewing tissue that requires a tightly regulated balance between keratinocyte proliferation and terminal differentiation to ensure proper structure and function. Despite substantial progress in characterizing the epigenomic and transcriptional regulatory mechanisms, the intricate control of epidermal homeostasis is still not fully understood. Emerging evidence is beginning to unveil the important role of post-transcriptional regulation by RNA-binding proteins, providing an additional layer of gene expression control essential for maintaining epidermal homeostasis and influencing skin tumor development. Cytoplasmic Polyadenylation Element Binding Proteins (CPEBs) are a family of four RNA-binding proteins (CPEB1-4) that regulate mRNA translation and stability by dynamically modulating the poly(A) tail length of specific mRNA targets. Although initially discovered during meiotic maturation, CPEBs have also been identified in somatic cells, extending their role beyond meiosis. However, CPEB1 and CPEB4 have not been examined together in the context of epidermal homeostasis, and their specific contributions to this tissue remain unknown. In this work, we have characterized the roles of CPEB1 and CPEB4 in keratinocytes and the interfollicular epidermis. Our findings reveal opposing functions of CPEB1 and CPEB4 in epidermal homeostasis and skin tumorigenesis. We have identified their mRNA targets in keratinocytes and uncovered a novel and contrasting role of CPEB1 and CPEB4 in keratinocyte differentiation. Both proteins are upregulated during keratinocyte differentiation and mediate a specific post-transcriptional control that fine-tunes cellular profiles toward either proliferation or differentiation. Notably, by focusing on the Notch signaling pathway, we found that the deletion of CPEB1 and CPEB4 in keratinocytes results in opposing patterns of Notch activation, which correlate with distinct keratinocyte differentiation states. Altogether, our results provide mechanistic insights into how CPEB1 and CPEB4 are involved in keratinocyte differentiation, potentially through the modulation of Notch signaling. This study further underscores the relevance of post-transcriptional regulation by RNA-binding proteins in maintaining tissue homeostasis and offers new perspectives on the role of CPEBs in skin biology.
Deciphering the importance of heterogeneity in Myotonic Dystrophy type 1
(Universitat de Barcelona, 2024-12-16) Núñez Manchón, Judit; Nogales Gadea, Gisela; Universitat de Barcelona. Facultat de Biologia
[eng] Myotonic dystrophy type 1 (DM1) is a genetic and multisystemic muscular dystrophy with autosomal dominant inheritance. It is caused by a CTG expansion in the 3’ end of DMPK gene. Symptoms are mainly caused by the accumulation of toxic RNA aggregates, called RNA foci, which sequester MBNL1 protein, a splicing regulator. The symptoms can appear at any age and they are highly heterogeneous between patients. This heterogeneity is partially derived from the CTG repeat length, which due to both somatic and intergenerational instability, differs both between patients and between different tissues of a single patient. In this thesis the main focus is put on DM1 heterogeneity, which is often ignored in studies. Heterogeneity is an important feature to consider because it could have an influence in the molecular DM1 alterations at cellular level and in the efficacy of a therapy, meaning that DM1 models representing said heterogeneity would be useful. The thesis is divided in three chapters, each of which, explores DM1 heterogeneity from a different perspective. Chapter 1 is focused on the study of heterogeneity from a molecular point of view. The study analyses differences in RNA foci and MBNL1 aggregates expression between different cell types or DM1 subtypes. On the one hand, the analysis of four different cell types (lymphoblasts, fibroblasts, myoblasts and myotubes) revealed significant differences between them, being myotubes the cell type with a higher number of RNA foci and myoblasts the cell type with a higher MBNL1 sequestration. Myoblasts were also used to perform a single cell study to determine heterogeneity in expression levels and whether it correlates with the number of RNA foci expressed in the same cell. Results revealed no correlation. On the other hand, the analysis of RNA foci in the 5 subtypes of DM1 (congenital, infantile, juvenile, adult and late-onset) revealed a higher accumulation of RNA foci in patients with an earlier age of onset. In this chapter the existence of molecular heterogeneity both between and within patients is confirmed. Chapter 2 is focused on how DM1 heterogeneity impacts the degree of efficacy of a treatment. The study assesses the therapeutic potential of antagomiRs 23b and 218, two antisense oligonucleotides that block MBNL1 repressors miRNAs 23b and 218. A previous study proved that these antagomiRs have a positive impact in DM1 pathogenesis, however DM1 heterogeneity was not assessed. In this study eight independent human DM1 primary myoblast lines with CTG repeats ranging from 117 to 1054 were treated with both antagomiRs. It was found that miR-23b and 218 are upregulated in patient myoblasts and that miRNAs overexpression correlated with CTG expansion size. The treatment reduced the miRNAs activity, increasing MBNL1 protein levels and reducing DMPK expression and foci number. Moreover, MBNL1- dependent splicing events were significantly rescued and the degree of MBNL1 enhancement correlated with splicing rescue. In this chapter it is concluded that antagomiRs 23b and 218 have therapeutic potential across different genetic backgrounds. Chapter 3 is focused on the need for in vitro models to study DM1 and to validate therapies that represent the clinical and genetic heterogeneity observed in DM1 patients. In the study, three DM1 muscle lines derived from patients with different DM1 subtypes and clinical backgrounds were immortalized and characterized at the genetic, epigenetic, and molecular levels. Results showed that the three immortalized cell lines displayed all the expected DM1 hallmarks with significant differences between the cell lines for several of the studied alterations. Moreover, the response of the immortalized cell lines to the previously tested therapeutics was also analysed with positive results. In this chapter it is concluded that the three immortalized DM1 cell lines developed in this study are suitable to study the pathophysiological heterogeneity of DM1 and to test future therapeutic options.
Towards a large-scale human inflammation atlas at single-cell resolution
(Universitat de Barcelona, 2024-12-12) Jiménez Gracia, Laura; Heyn, Holger; Gut, Ivo G.; Universitat de Barcelona. Facultat de Biologia
[eng] Inflammation is a physiological response essential for maintaining homeostasis, but when the immune system becomes dysregulated, it can drive a range of pathological conditions and become a key contributor to many diseases. Understanding these complex, disease-specific inflammatory mechanisms is crucial for developing effective treatments. Single-cell sequencing technologies offer a powerful approach to study the immune system at high resolution, providing detailed insights into cellular heterogeneity and immune dynamics. To fully exploit their potential in large-scale studies, standardized protocols for sample collection and processing, coupled with advanced computational methods for robust data analysis, are needed. Since immune-driven diseases often target specific tissues and exhibit distinct inflammatory profiles, a comprehensive approach is required. Blood, as a minimally invasive sample, offers a valuable opportunity to monitor inflammation by tracking cytokine levels and cellular activity, making it ideal for elucidating disease mechanisms and therapeutic responses. In this thesis, experimental protocols and computational approaches were developed for large-scale studies focused on analyzing human inflammatory profiles using single-cell RNA sequencing (scRNA-seq). First, we implemented FixNCut, a protocol that preserves transcriptional profiles and tissue composition by fixing samples at the time of collection, enabling subsequent tissue dissociation with minimal stress-induced artifacts. We then generated a comprehensive reference of the inflammatory spectrum in circulating immune cells using single-cell transcriptomic profiles from over 1,000 patients suffering from 20 immune-driven diseases and healthy individuals. By analyzing the immune cell composition and inflammatory molecule expression, we characterized the immune response across diseases, identifying both commonalities and disease-specific differences. Using interpretable machine learning algorithms, we captured disease-specific inflammatory genes at the cell type level that could serve as clinical biomarkers. Furthermore, we proposed a framework for classifying patients based on their blood-derived inflammatory profiles. Finally, we studied the immunomodulatory effects of albumin treatment in patients with acute decompensated cirrhosis, which resulted in immune transcriptional reprogramming that primarily affected B lymphocytes and enhanced neutrophil antimicrobial activity, boosting the patients' immune response against infections. In summary, this thesis integrates scRNA-seq experimental and computational methods to facilitate large-scale, decentralized research on immune-driven diseases with the goal of applying these approaches for disease diagnosis, monitoring progression, and assessing therapy responses in clinical settings.
Enhancing variant classification in hereditary breast and ovarian cancer predisposition genes through splicing analysis and new tumour evidence
(Universitat de Barcelona, 2024-12-09) Domènech Vivó, Joanna; Gutiérrez Enríquez, Sara; Díez Gibert, Orland; Universitat de Barcelona. Facultat de Biologia
[eng] The clinical management of hereditary breast and ovarian cancer (HBOC) families relies on identifying germline pathogenic variants in cancer susceptibility genes, such as BRCA1 and BRCA2. However, genetic testing often yields an inconclusive result due to detection of only neutral variants or to the identification of variants of unknown significance (VUS), whose effects on gene function and associated cancer risk are unknown. While carriers of pathogenic variants benefit from options available for early cancer detection, preventive measures and targeted therapies, VUSs carriers are generally managed without regard to the result of genetic testing, thus limiting the options for these patients. The primary aim of this thesis is to increase the number of patients receiving a genetic diagnosis of HBOC by improving the accuracy of classifying germline genetic variants. To achieve this, we focused on improving the detection of potentially splicing-altering variants in poorly understood deep intronic regions (Part 1), evaluating the relevance of mRNA splicing experimental validation for variant classification (Part 2) and exploring the potential use of tumour information in the classification of germline variants (Part 3). Deep intronic regions may contain variants that affect RNA splicing by introducing “pseudoexons” or other changes, which could potentially explain cases where the genetic cause of HBOC remains unknown. Current computational in silico tools to predict spliceogenic variants leading to pseudoexons have limited efficiency. In Part 1 of this thesis, we have assessed the performance of the SpliceAI tool combined with ESRseq scores to identify spliceogenic deep intronic variants by affecting cryptic sites or splicing regulatory elements (SREs) using literature and experimental datasets. Our results with 233 published deep intronic variants showed that SpliceAI, with a 0.05 threshold, predicts spliceogenic deep intronic variants affecting cryptic splice sites, but is less effective in detecting those affecting SREs. Next, we characterised the SREs profiles using ESRseq, showing that pseudoexons are significantly enriched in SRE-enhancers compared to adjacent intronic regions. This indicates that intronic regions with a high potential to be included as pseudoexons can be systematically identified throughout the HBOC genes. Although the combination of SpliceAI with ESRseq scores (considering ∆ESRseq and SRE landscape) showed higher sensitivity, the global performance did not improve because of the higher number of false positives. Both tools combined were tested in a tumour RNA dataset with 207 intronic variants disrupting splicing, showing a sensitivity of 86%. Following the pipeline, five spliceogenic deep intronic variants were experimentally identified from 33 variants in HBOC genes. Overall, our results provided a framework to detect deep intronic variants disrupting splicing. A possible pathogenic mechanism for a subset of VUS is the disruption of normal mRNA splicing. However, experimental validation is generally restricted to variants affecting dinucleotide splice site positions. Consequently, variants outside these regions are not consistently evaluated across laboratories, leading to conflicting and uncertain interpretations. Additionally, variants leading to in- frame isoforms affecting clinically relevant domains are challenging to classify due to the limited knowledge about in-frame encoded proteins. One clear example of both issues are the variants located in the in-frame exon 18 (legacy number) of BRCA1. The results of a comprehensive study conducted within the ENIGMA consortium to identify spliceogenic variants mapping to this exon and characterize the severity of their impact are presented in Part 2 of this thesis. Patient RNA, splicing reporter minigene, and mouse embryonic stem cell (mESC)-based functional assays were combined to determine the impact of 166 variants on splicing and estimate the tolerability functional range of exon 18 skipping (∆18). Splicing, functional and clinical data were integrated for variant interpretation according to the recently ClinGen-approved BRCA1/BRCA2- specified ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular-Pathology) classification guidelines. Our results demonstrated that exon ∆18 is not a rescue event and that per-allele levels up to 34%, combined with at least 59% of the full-length (FL) isoform, are tolerable. Furthermore, we show that applying the recently developed BRCA1/2 specifications significantly reduced the rate of VUS in this exon from 45.7% to 8.5%, being experimental mRNA testing essential in solving uncertainty for approximately 10% of variants Lastly, despite the existence of multiple types of evidence for classifying germline variants, additional information is still required to classify many of them. Accordingly, we explored the potential use of tumour homologous recombination repair (HRR) status by RAD51 foci immunofluorescence detection (RAD51 test) as a predictor of pathogenicity or benignity for BRCA1/2 and PALB2 germline variants. HRR status by RAD51 test was successfully assessed in a total of 148 primary untreated breast and ovarian tumours, from Vall d’Hebron Hospital, ENIGMA and EraPerMed RAD51predict collaborators. Samples were split into three cohorts depending on patients’ germline status: pathogenic (91), benign (47) and VUS (8). Our results demonstrated that the RAD51 test could distinguish pathogenic from benign variants with high sensitivity (91.5%) and specificity (70.2%) (AUC 0.91). Likelihood ratios (LR) were calculated for the association of RAD51 test outcomes (HRR deficiency-HRD or proficiency-HRP), with BRCA1, BRCA2 and PALB2 variant pathogenicity considering patients' clinical features. Then LR estimates were aligned to ACMG/AMP code strengths. Overall, RAD51-HRD status provided moderate evidence for pathogenicity if detected in primary breast tumours diagnosed before age 50, whereas RAD51-HRP provided informative moderate evidence against pathogenicity if detected in primary breast tumour independently of age at diagnosis. Lastly, we applied our recommendations to classify the eight VUS and about 38% (3/8) of variants were classified as likely benign, thus demonstrating the utility of the RAD51 test in variant classification.
Monocytes and macrophages in cancer: investigating their potential as disease biomarkers and therapeutic targets
(Universitat de Barcelona, 2024-12-02) Paul, Tony; Sarrias Fornés, Maria Rosa; Universitat de Barcelona. Facultat de Biologia
[eng] Cancer is the leading cause of death worldwide, with lung and liver cancers being major contributors. Liver cancer is often preceded by cirrhosis, considered as the final stage of chronic liver disease. Recognizing the crucial role of the immune system in cancer, ongoing research focuses on improving immunotherapy effectiveness to benefit a broader patient population. Circulating monocytes and tumor-associated macrophages (TAMs) play key roles in the immune response, contributing to tumor progression, treatment outcomes, and survival in various cancers, including lung and liver. TAMs are the most abundant immune cells in the tumor microenvironment (TME). They can adopt either a pro-inflammatory (M1) or immunosuppressive (M2) phenotype, with the M2 correlating with poorer survival outcomes in cancers. Current research is exploring TAM reprogramming as a novel cancer immunotherapy, with monoclonal antibodies offering safer and more effective alternatives to chemotherapy. On the other hand, monocytes are promising biomarker candidates due to their diverse phenotype and accessibility. The two main objectives of this thesis were to investigate the role of macrophage secreted protein CD5L expression in TAMs in lung and liver cancers, contributing to the development of a new CD5L-targeted immunotherapy, and to analyze the proteome of circulating monocytes in cirrhosis and liver cancer to identify new biomarkers and altered biological pathways occurring in hepatocellular carcinoma (HCC). We show that CD5L expression in TAMs is linked to poor prognosis in papillary lung adenocarcinoma (PAC) and HCC. Additionally, targeting CD5L in a mouse model reduced tumor size and reprogrammed the TME into a pro-inflammatory state. We also discovered deregulated pathways affecting monocyte activity in cirrhosis and HCC, and identified a new cell receptor panel as a potential blood-based biomarker for HCC. These findings mark significant progress in creating a novel anticancer immunotherapy that induces beneficial changes in the TME and identifying a promising biomarker for HCC detection.
Spatiotemporal Changes in Mountain Plant Communities of the Central-Eastern Region of the Trans-Mexican Volcanic Belt Over the Last 17,000 Years
(Universitat de Barcelona, 2024-11-26) Rodríguez Pérez, Erandi Tzayani; Montoya Romo, Encarnación; Universitat de Barcelona. Facultat de Biologia
[eng] Neotropical mountains are known for their high biodiversity and endemism, providing numerous ecosystem services to the surrounding areas. However, mountain biodiversity is rapidly declining due to climate change and anthropogenic activities. The present thesis uses pollen analysis to investigate changes in high mountain neotropical plant communities in the central-eastern region of the Trans- Mexican Volcanic Belt (TMVB), in Central Mexico, over the past 17 kyr. Two study sites have been integrated into this thesis: Cofre de Perote and Iztaccíhuatl volcanos. At an elevation of 3700 m asl, the pollen assemblage of a sedimentary record from Cofre de Perote volcano was analysed, supported by charcoal, Magnetic Susceptibility (MS), and Ti concentration analyses. This research documented the vegetation reorganisation and plant colonisation of high elevations after the glacial retreated due to temperature increase during the Early and Mid-Holocene. The results suggested that during the Early Holocene, forests were maybe closer and/or at higher elevations in Cofre de Perote, near the coast, than at similar elevations in inland sites. The results also revealed that the influence of winds on long-distance pollen arrival could play a crucial role in understanding the pollen assemblages in highland sites. Additionally, Picea pollen presence indicated the occurrence of non-analogue communities in this site and its potential role as microrefugia during the Early Holocene. In the Iztaccíhuatl volcano, the pollen deposition in surface soil samples from the current plant communities along an elevation gradient was analysed. The results suggested that high percentages of Abies were related to the presence of a Fir Forest. High Pinus values and Arceuthobium pollen presence could be associated with Pine Forest. Pollen taxa such as Apiaceae, Eryngium, Valerianaceae, and Caryophyllaceae are linked to Pine Forest and Alpine Grassland. Secondly, the research focused on studying past plant communities' distribution along the volcano's elevation gradient via pollen analysis of palaeosol samples from three elevation transects. The samples collected in each elevation transect spanned a specific age (time window) during the Late Quaternary. The recovery of samples was made using three tephras that potentially sealed the soil protecting the palynomorphs from degradation, and at the same time, worked as chronostratigraphic markers for this research: Tutti Frutti (~17 cal kyr BP), Ochre (~5.7 cal kyr BP), and Pink (~1.1 cal kyr BP/840 CE) Pumices. In addition, the eruption events resulting in the tephra deposits occurred coeval to three distinct climatic events: Heinrich Event-1 (HE-1), Holocene Thermal Maximum (HTM), and Medieval Climatic Anomaly (MCA). Results indicated that samples at ~17 cal kyr BP lack sufficient pollen grains to propose an accurate paleoenvironmental interpretation. At ~5.7 cal kyr BP, high Poaceae pollen values suggested the extensive presence of open vegetation. At ~1.1 cal kyr BP, the presence of Mixed Forest between ~3300 and 3450 m asl, Pine Forest between 3450 and 3700 m asl, and a more extended ecotone zone between Pine Forest and Alpine Grassland was proposed. Inferred plant community distribution and structure changes at ~5.7 and ~1.1 cal yr BP may be linked to warm and dry conditions during HTM and MCA, which seemed to have a larger effect on trees than on herbs. The join interpretation of this thesis has revealed the vulnerability of coniferous forests to global warming and human activities. These past and current threats could be involved in: (i) a possible recent expansion of Fir Forest around the Iztaccíhuatl volcano and other areas of the TMVB; and (ii) the evidence of Picea local extinction in TMVB during the Early Holocene, suggesting that species with restricted distributions or isolated populations are at risk. Finally, the importance of natural reserves and conservation programs is highlighted.
Characterization of maternal histone mRNAs
(Universitat de Barcelona, 2024-11-29) Pérez Roldán, Juan Francisco; Azorín, F.; Carbonell Sanroma, Albert; Universitat de Barcelona. Facultat de Biologia
[eng] In eukaryotes, histone proteins bind and pack genomic DNA into chromatin. The basic structural subunit of chromatin is the nucleosome, which is formed by the interaction of an octamer of the core histone proteins H2A, H2B, H3 and H4 with 147 bp of DNA. In addition, linker histones H1 bind to the nucleosome core particle at the entry/exit site of nucleosomal DNA and interact with the internucleosomal linker DNA. According to their pattern of expression during the cell cycle, histones are classified into replication dependent (RD), whose expression is tightly linked to DNA replication, and replication independent (RI). RD histones include the four canonical core histones and most linker histones H1 and are involved in packaging the newly generated DNA during replication. On the other hand, histone variants are generally RI. RD histone mRNAs are distinct from every other mRNA in the cell since they are not polyadenylated. Instead, they have a conserved sequence forming a stem-loop at the 3’UTR, which is recognized by the Stem-Loop Binding Protein (SLBP). SLBP is responsible for processing, stabilization, and translation of RD histone mRNAs, as well as for cell cycle coordination of their expression. In contrast, RI histone mRNAs are polyadenylated. In metazoans, early embryogenesis usually involves rapid nuclear divisions in the absence of zygotic expression. The number and speed of these divisions are generally higher in species with external development. In Drosophila, there are 13 syncytial nuclear divisions before zygotic genome activation (ZGA). During these early divisions, dSLBP is absent, and packaging of the rapidly generated DNA relies on maternal histones. Core RD histones are maternally deposited as both proteins and mRNAs. Instead, we found that the single RD linker histone of Drosophila, dH1, is deposited only as mRNAs. In addition to dH1, Drosophila encodes for a germline-specific linker histone dBigH1 variant, which is maternally deposited as both protein and mRNA. In this study, we show that, in Drosophila, maternally deposited RD histone mRNAs are polyadenylated and have a truncated 3’UTRs that disrupts the characteristic 3’ stem-loop structure. We also show that these unusual RD histone transcripts are generated during oogenesis, in stage 10 egg chambers, through an alternative processing that surprisingly requires dSLBP and involves cytoplasmic polyadenylation. Polyadenylation of maternal RD histone mRNAs does not appear to be restricted to Drosophila since it is also observed in Xenopus, which also undergoes external development. Intriguingly, we found that maternal RD histone mRNAs remain largely untranslated during early embryogenesis. In particular, despite being maternally deposited as mRNAs, expression of the single RD linker histone of Drosophila, dH1, is not detected during the early embryo divisions. Instead, at these stages, the germline-specific linker histone dBigH1 variant is expressed. dH1 expression starts at nuclear division 6 and increases progressively during ZGA, fully replacing dBigH1 at cellularization. Notably, we found that loss of dBigH1 in homozygous null bigH1 mutant embryos induces the early expression of dH1 from the first nuclear division, which is concomitant to increased translation of maternal dH1 mRNAs. Interestingly, homozygous null bigH1 mutant embryos progress normally through development. These results unveil the compensatory expression of dH1 in the absence of dBigH1, supporting functional redundancy of dBigH1 and dH1 during early embryogenesis. Finally, we show that translation of the rest of maternal RD histone transcripts is also increased in the absence of dBigH1. Altogether, these results suggest that translation of the maternal pool of RD histone transcripts serves as a backup mechanism that is induced in response to reduced supply of maternal histone proteins.
WNT pathway regulation of lipid handling and inflammation in organ function: the role of LRP5
(Universitat de Barcelona, 2024-10-09) Luquero Gomez, Aureli; Borrell Pagès, Maria; Badimón, Lina, 1953-; Universitat de Barcelona. Facultat de Biologia
[eng] INTRODUCTION: cardiovascular diseases are the first cause of premature mortality worldwide. Atherosclerosis is the most common underlying cause of cardiovascular disease. This is a complex condition triggered by the accumulation of modified cholesterol in the vascular wall, which induces inflammatory and fibrotic processes. Hypercholesterolemia is the elevation of circulating cholesterol levels in blood, which affects cholesterol homeostasis in different tissues and organs. Canonical WNT pathway is an evolutionary conserved signalling pathway with a role in embryonic development and cell fate. Low-density Lipoprotein Receptor – related protein 5 (LRP5) is a receptor that triggers the canonical WNT signalling and uptakes circulating low-density lipoproteins (LDL). It is our hypothesis that LRP5 and the canonical WNT signalling play a key role in the cellular response to cholesterol exposure in different tissues. OBJECTIVES: the aim of this thesis is to identify the role of LRP5 in different cell types and tissues modulated by hypercholesterolemia including circulating macrophages and cell lineages of the liver and the adipose tissue. Additionally, this thesis focuses on the study of LRP5 roles during the inflammatory response induced by atherosclerosis and its role in the brain (an organ with a heavily regulated cholesterol homeostasis).
Exploring the chromatin landscape and gene expression mechanisms
(Universitat de Barcelona, 2024-10-15) Sala Huerta, Alba; Orozco López, Modesto; Battistini, Federica; Universitat de Barcelona. Facultat de Biologia
[eng] The interplay between chromatin, transcription factors and genes defines a complex gene regulatory system whose study is essential to understand cell differentiation and how relevant cell functions are maintained or disrupted during biological processes. This is the main area of interest of what is known as 4D genomics, a field that has been evolving on the wave of high-throughput methods producing extensive amounts of data on gene location, chromatin structure and gene expression. 4D genomics data is each day more accessible, the challenge being to interpret it. In this sense Machine Learning (ML) and Artificial Intelligence (AI) methods are becoming crucial to transform noisy experimental data into biological information. The aim of this thesis is to untangle some of the mechanisms that constitute regulatory networks while integrating various state-of-the-art techniques to further understand some of the currently unanswered challenges. During my PhD thesis I have been exploring the chromatin landscape and gene expression mechanisms at different levels of detail and this volume summarizes the main results obtained. In the Introduction, a brief overview of gene expression and transcriptional regulation mechanisms is provided. I also discuss the underpinnings of chromatin organization and stress conditions. Chapters 1 through 5 are a compendium of articles where I describe the different research projects that I undertook during my PhD thesis. These projects have either been published in peer-review journals, are currently under review or in preparation. More specifically in Chapter 1 we started by studying the first regulatory layer, the double-stranded helical structure of DNA and its binding to effector proteins. We developed a ML model that predicted with high accuracy the in vitro affinities and binding sites of various transcription factors based on physical properties of the DNA. Our method also successfully reproduced in vivo data when combined with a second layer of information, the chromatin organization of the nucleosomes. In Chapter 2 we explored nucleosome positioning preferences in yeast genomic DNA by first developing a predictor of nucleosome free regions around the transcription start and terminating sites which are known to comprise critical binding sites. Our method allowed us to predict the nucleosome architecture within gene bodies by using signal theory from two strongly positioned nucleosomes referred to as +1 and -last (the nucleosomes immediately downstream of the TSS or upstream of the TTS respectively). We additionally studied the link between nucleosome arrangements and gene expression mechanisms. In Chapter 3, the effects of oxidative stress damage on nucleosome organization and overall chromatin structure are described. In order to clarify the effect of these lesions, we performed statistical analysis on a series of gene expression mechanisms through different experimental techniques such as MNase-Seq, Hi-C and Micro-C experiments. Chapters 4 and 5 introduce the study of RNA as a distinct structure and discuss its properties and capability of playing a key role in some regulatory mechanisms such as triplex forming oligonucleotides. A general discussion that encompasses the significance and future perspectives of these 5 projects is presented in Chapter 6, together with the main conclusions of this work in Chapter 7.
Genomic applications to the study of Western Mediterranean amphibians and reptiles, with a focus on the genera Calotriton, Iberolacerta, and Vipera
(Universitat de Barcelona, 2024-10-25) Talavera Payán, Adrián; Carranza Gil-Dolz del Castellar, Salvador; Universitat de Barcelona. Facultat de Biologia
[eng] The advent of genomics has revolutionized the study of reptiles and amphibians at different levels and fields. In particular, this thesis deals with the applications of genomics to systematics, conservation and evolutionary biology basic research, focused on three Western Mediterranean genera –Calotriton, Iberolacerta, and Vipera–. Regarding the brook newts of the genus Calotriton, reduced-representation genomic analyses revealed severe bottlenecks triggered by climate changes, especially in the Montseny brook newt (C. arnoldi), the most threatened amphibian species in Europe, that led to depauperate genome-wide heterozygosity values and high fragmentation among populations. Populations in the Western Montseny submassif seemed to be significantly more different than expected, and backed by genomic, morphological and ecological evidence, we formally described the Western Montseny brook newt (C. arnoldi laietanus) as a new subspecies, aiming to highlight the uniqueness of this lineage and ensure appropriate management and protection of this Critically Endangered taxon. Secondly, rock lizards from the genus Iberolacerta served as model to explore major rearrangements in genomic architecture, potentially linked to ecological adaptation. We found support for two alternative hypotheses explaining the alpine confinement of this genus using whole-genome sequencing data. On the one hand, demographic inference supported ecological outcompetition reflected by major bottlenecks coinciding with the radiation of the genus Podarcis, and, on the other, adaptations to altitudinal hypoxia were found in the form of gene expression shifts on hemoglobin isoforms, which might render toxic higher oxygen concentrations. In third place, the European vipers of the genus Vipera exhibited the most conspicuous patterns of mito-nuclear discordance in their phylogeny, which is, moreover, pervaded by introgression among old-diverged species. Population genomics in Iberian vipers unveiled evidence for adaptive introgression among an ecologically dissimilar species pair, whereas chromosomal rearrangements have enabled ecological convergence between a sympatric and ecologically similar species pair. Finally, we characterized the venom-encoding genes of the three Iberian vipers coupling genomic, transcriptomic, and proteomic evidence to reveal a fine balance between opposing selective forces governing the evolution of venom as an integrated phenotype. Overall, this thesis underscores the role of genomics as the new cornerstone of modern systematics, an ideal instrument for guiding conservation programs, and a powerful tool to unravel key aspects and drivers of speciation, adaptation and other evolutionary processes.
Next Generation Sequencing in Early-Onset Parkinson’s Disease: Insights into the Role of Endo-Lysosomal Pathway from Genetic Knowledge
(Universitat de Barcelona, 2024-10-25) Pascual Rodríguez, Alba; Hoenicka Blanco, Janet; Palau Martínez, Francesc; Universitat de Barcelona. Facultat de Biologia
[eng] Parkinson’s Disease (PD) is the second most common neurodegenerative disorder, with an etiology remaining unknown in the majority of patients. From a genetic perspective, PD is classified into Mendelian cases (15%) and sporadic cases (85%). The genetic architecture of sporadic cases is complex due to risk factors with incomplete penetrance that could facilitate the expression of PD, alongside gene-environment interactions. PD exhibits considerable heterogeneity, and the etiological diagnosis of the disease remains elusive in many cases. In the study of Mendelian forms of PD, it has been possible to identify genes involved in diverse biological pathways, including vesicular trafficking, autophagy, and dysfunction of organelles such as lysosomes and mitochondria. The aim of this study was to identify new variants that could potentially cause or increase the risk of PD through genomic studies. These variants may follow a Mendelian inheritance pattern or contribute to an oligogenic inheritance, affecting one or several pathways involved in the pathogenic mechanisms of PD. Notably, there is evidence of a greater contribution of genetic factors in early- onset Parkinson's Disease (EOPD); hence, our investigation was conducted in this group of patients. One approach implemented in this study was the analysis of variants in specific regulatory regions of PD-associated genes. Among the five non-coding candidate variants identified, the c.-253C>T variant in the 5'UTR of the DNAJC13 showed an allele-specific impact. Functional studies indicated a significant effect on gene expression and basal autophagy activity in the patient's fibroblasts. This variant affects the regulation of DNAJC13 expression, suggesting possible dysfunctions in vesicular transport and autophagic processes. The application of whole exome sequencing (WES) in a cohort of 49 patients enabled the genetic diagnosis of 9 patients (18.36%) through a knowledge-driven analysis designed in our laboratory that prioritises filtered variants. Additionally, the WES data allowed us to develop a new biological approach, considering aspects such as the molecular mechanisms underlying PD and the subcellular location of proteins to subgroup patients based on their most affected pathways. This multifaceted approach highlighted the relevance of variants in lysosomal genes, such as GLA:p.Asp313Tyr and GLB1:p.Arg419Gln. Functional studies on the patients’ fibroblasts showed deficiencies in lysosomal enzymes and alterations in the morphology of the Golgi apparatus and lysosomal network, suggesting that the lysosomal dysfunction could contribute to the etiology of EOPD patients. The identified variants through WES suggest an oligogenic mode of inheritance for EOPD, where the combination of variants can be unique for each EOPD patient. We also evaluated a new allele, LRRK2:p.Leu119Pro;p.Leu488Pro in a PD-affected family. This new allele is proposed as the cause of the disease in the family. Although the change did not affect the function, expression and stability of the protein, the increased interaction of the mutated LRRK2 with Rab8a suggests an impairment in vesicular trafficking. These findings emphasize the importance of endo-lysosomal pathway dysfunction in the etiology of EOPD, and highlight the need for new perspectives in genomic data interpretation to understand the genetic architecture of this heterogeneous disease.
Molecular characterization of the histone demethylase PHF2 in maintaining proliferation, homeostasis and genomic integrity of neural progenitors
(Universitat de Barcelona, 2024-09-10) Aguirre Infantes, Samuel; Martinez Balbas, Mª Angeles; Universitat de Barcelona. Facultat de Biologia
[eng] The histone demethylase PHF2, previously studied in the laboratory, has been shown to be crucial for the expansion of neural progenitors by maintaining low levels of H3K9me2/3 at the promoters of cell cycle and developmental genes. It was also discovered that the absence of PHF2 induces the expression of repetitive elements such as Major Satellite, and that PHF2 interacts with components of heterochromatin. The thesis focuses on the interaction of PHF2 with constitutive pericentromeric heterochromatin and its role during mitosis. Experiments were conducted on neural stem cells (NSCs) both in control conditions and depleted of PHF2 through shRNA. The effects were analyzed across three areas of chromatin: euchromatin, pericentromeric heterochromatin (PcH), and the borders of PcH. In euchromatin, PHF2 was observed to localize at the promoters of cell cycle and developmental genes. Depletion of PHF2 resulted in increased levels of H3K9me3 and decreased accessibility at these promoters. In heterochromatin, PHF2 was enriched in satellite repeat sequences, such as GSAT_MM. Depletion of PHF2 increased the accessibility of heterochromatin and decreased H3K9me3 levels, leading to increased transcription of repetitive sequences. Live imaging experiments confirmed these changes in methylation. Upon observing the disorganization of heterochromatin and considering its nucleation and extension mechanisms described in the literature, the study examined the boundary region between heterochromatin and euchromatin to elucidate what was causing this disruption of the heterochromatin and further genome instability. A significant enrichment of PHF2 was observed at the borders of pericentromeric heterochromatin (from megabase 3 to 3.5 on each chromosome). Depletion of PHF2 in these regions led to an increase in H3K9me3 and a decrease in accessibility, suggesting that PHF2 balances H3K9me3 across the genome. Eliminating PHF2 causes an imbalance between euchromatin and heterochromatin, silencing euchromatic regions and de-repressing previously repressed genes that were not targeted by PHF2. The integrity of heterochromatin can be affected either by the lack of transcriptional activity in the boundary regions or by the dilution of constitutive heterochromatin components, which are limited. This increases the expression of repetitive sequences, causing genomic instability and DNA damage. PHF2 requires both its PHD and JmjC domains to maintain the balance of H3K9me3 and genomic integrity. This was confirmed through overexpression and rescue experiments with mutants for each of these domains in shPHF2 neural stem cells. The positively charged amino acid-rich domain, important for phase separation and gene expression, is not essential for the integrity of heterochromatin. In collaboration with Amanda Fisher's laboratory at the University of Oxford, the role of PHF2 during mitosis was studied. It was observed that PHF2 binds to mitotic chromosomes, affecting chromosomal size: depletion of PHF2 reduces chromosome size and alters methylation marks such as H3K9me3 and H3K27me3. An increase in H3K27me3 in the body of the chromosome and a reduction in H3K9me3 levels in the chromosome centromere were observed. Temporal transcriptomics revealed that post- mitotic transcription reactivation was delayed when PHF2 was depleted, especially in histone genes. This doctoral thesis has significantly advanced the understanding of PHF2's role in balancing H3K9me3 and maintaining genomic stability. The research has demonstrated the importance of PHF2 in chromatin regulation and the proliferation of neural progenitor cells, highlighting its critical function in both heterochromatin and mitosis.
Targeting the TGF-ß pathway, SMAD proteins and cofactors
(Universitat de Barcelona, 2024-07-10) Torner Blancafort, Carles; Macías Hernández, María J.; Martínez, Aurora; Universitat de Barcelona. Facultat de Biologia
[eng] TGF-ꞵ signaling is key for many biological processes as embryo development, tissue homeostasis and immune system regulation. When altered, this pathway can lead to diseases such as cancer, fibrosis and rare syndromes. Key elements of the pathway are the SMAD family of transcription factors, which translate the extracellular signal received by the TGF-ꞵ receptor to the nucleus for regulation of gene expression. SMAD proteins have a characteristic structure which is shaped by an MH1 domain, for specific DNA recognition, a flexible linker region, and their MH2 domain, which can form complexes with other SMAD proteins and co-factors. This last domain is often mutated in disease, especially in the case of SMAD4 for which single point mutations and deletions have been identified in the literature. In this work, I focused on the study of SMAD4 variants associated with diseases, such as cancer, Juvenile Polyposis Syndrome, Hemorrhagic Hereditary Telangiectasia and Myhre Syndrome. With this aim, we produced different recombinant protein constructs to study the effect of these variants in their fold and binding properties. Firstly, I started with the characterization of the variants R496C- and I500V/M/T- SMAD4, associated with Myhre Syndrome. This is a gain-of-function disease that begins during embryonic development, and the alterations observed lead to the dysfunction of multiple organs. We could confirm that these specific SMAD4 variants had increased levels of SMAD4 protein in cells, possibly related to decreased ubiquitination and degradation of the protein, among other possible causes are loss-of-function variants, as in gastrointestinal cancers and Juvenile Polyposis. In this case, our work showed that the complexes with R-SMADs and the variants lead to several different stoichiometries compared to those of the wild type (WT) protein. The second section of this thesis is focused on the search for small-molecules as SMAD4 binders. We used single molecule biophysics and structural biology to identify pharmacological strategies based on targeting SMAD4 to modulate TGF-ꞵ signaling. This search was conducted through a target-based in vitro approach using purified SMAD4 MH2 domain and large libraries of compounds. Among these compounds, we included FDA-approved drugs in case we could identify hits that could be repurposed to treat individuals suffering from very rare syndromes. Validated hits have affinities of interaction ranging between low and high micromolar and will be further developed and tested. Some interesting. approved drugs were identified as SMAD4 binders. In the last chapter of this project, I focused on the DNA recognition ability of Ras Responsive Element Binder 1 (RREB1). RREB1 plays a key role in communication between RAS and TGF-ꞵ signaling to regulate epithelial-to-mesenchymal transition (EMT) during embryonic development and maintenance of healthy tissue, but also during cancer progression. RREB1 is a zinc finger (ZF) protein with multiple isoforms. In particular, I studied a well-conserved evolutionary ZF pair located at the C-terminus of the protein.
Anomalous diffusion and search behavior in Caenorhabditis elegans
(Universitat de Barcelona, 2024-06-20) Lloret Cabot, Roger; Bartumeus Ferré, Frederic; Universitat de Barcelona. Facultat de Biologia
[eng] In this thesis, our focus was on unraveling how Caenorhabditis elegans samples its environment in the absence of gradients and food resources. We analyzed data from a relocation experiment in which individual worms, including wild-type isolates and defective mutants, were displaced from a food patch to an empty arena in tightly controlled environmental conditions. We characterized the spreading patterns of the worms and analyzed their movement dynamics at various temporal scales, ranging from milliseconds to hours. Our findings revealed an intricate, time-dependent spreading process, likely driven by internal states and memory relaxation due to the absence of clear external stimuli. Notably, C. elegans exhibited superdiffusive spreading behavior, prompting us to investigate the mechanisms behind this phenomenon and its adaptive significance in search contexts. To this end, we devised a classification algorithm that enabled us to characterize C. elegans trajectories into its elementary motor behaviors: sharp turns and crawls. Leveraging these insights, we developed a mechanistic model of movement that reproduces C. elegans search trajectories in great detail, shedding light on the emergence of superdiffusion. Additionally, we evaluated the search performance of various C. elegans-inspired movement models across multiple spatial scales, assuming two distinct search conditions: symmetric (all targets distant) and asymmetric (near and far targets) scenarios. Through this exercise, we quantitatively assessed the contribution of both superdiffusion and turning dynamics to the search performance of C. elegans. Finally, in an effort to reinforce our theoretical results with a real case study, we conducted a series of experiments with C. elegans individuals foraging in patchy landscapes with bacteria. In these experiments, we employed both homogeneous (regular) and heterogeneous (aggregate) landscapes, mirroring the search conditions evaluated in the synthetic simulations. Our findings confirmed that turning dynamics and superdiffusion are key elements of search behavior in C. elegans, fundamentally shaping its search performance in all landscape configurations analyzed. Furthermore, our results suggest that these two mechanisms have the potential to influence the ecological fitness of the species in the natural environment. While our conclusions are primarily based on the specific experimental conditions analyzed, which involve a sharp transition from resource-plentiful to empty arenas, we believe that the findings we present offer valuable insights into both the understanding of C. elegans search behavior and the adaptive value of superdiffusion and turning dynamics in search processes.

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