Tesis Doctorals - Facultat - Medicina

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Integrative Chromatin Profiling in Mature B cell Lymphomas: Biological and Clinical Insights
(Universitat de Barcelona, 2024-04-26) García Torre, Beatriz; Martín-Subero, José Ignacio; Kulis, Marta; Universitat de Barcelona. Facultat de Medicina
[eng] The DNA molecule carries all the genetic information necessary for the development of living organisms. DNA is compacted in nucleus of human cells, wrapped around the nucleosome, a core of proteins called histones. The organization of the nucleosomes along the DNA molecules and the post translational modifications that histones may have, can influence gene expression. Study of epigenetic marks, such as DNA accessibility and histone modifications, can expand our knowledge on the biology of certain diseases such as lymphomas. In this doctoral thesis, different histone marks, chromatin accessibility and transcriptomics have been integrated to characterize the role of the chromatin modulation in the biology of mantle cell lymphoma (MCL) and Richter transformation (RT) of chronic lymphocytic leukaemia (CLL). I initially generated 10 MCL reference epigenomes that include six histone modifications, chromatin accessibility and gene expression (Study 1). An integrative analysis of these epigenomic layers revealed an MCL-specific chromatin signature as compared to normal B cells. The de novo active regulatory regions identified were linked to a set of genes related to cell development and activation. The two described MCL subtypes, conventional (cMCL) and the leukemic non-nodal MCL (nnMCL), show biological, genetic, transcriptomic, and clinical differences. I observed that they also present differences at the chromatin level. The analysis of cMCL specific regulatory regions revealed an enrichment in specific transcription factor families responsible for transcription regulation of genes involved in proliferation and carcinogenesis (i.e. Krüppel-related, ETS-related, BHLH-ZIP, Jun-related, E2F, and CEBP-related TF families). On the contrary, nnMCL did not show major changes in chromatin activation as compared to cMCL, showing relatively minor differences to normal B cells. Moreover, I identified that the different levels of chromatin activation among cMCL samples are related to clinical behaviour, with more activation associated with shorter overall survival (Study 2). Furthermore, chromatin analyses were integrated with genome and transcriptome to characterized Richter transformation (RT), an aggressive evolution of CLL into an aggressive large B cell lymphoma. We found small subclones carrying genomic, and transcriptomic features of RT cells already at CLL diagnosis, which were dormant for up to 19 years before transformation. We also identified new driver alterations and discovered a new mutational signature (SBS-RT). Analysing the chromatin landscape, I identified patterns of both chromatin activation (H3K27ac) and chromatin accessibility (ATAC-seq) unique for RT. Integration of epigenetics and transcriptomics revealed an oxidative phosphorylation (OXPHOS)high–B cell receptor (BCR)low-signalling transcriptional axis in RT, which has potential therapeutic implications (Study 3). In summary, this doctoral thesis provides significant insights into the role of the chromatin landscape in the biology of mature B cell lymphomas, showing that chromatin activation is linked to disease aggressiveness.
Regulation of CK2 by phosphoinositides and inositol pyrophosphates: implications in endocytosis
(Universitat de Barcelona, 2024-02-01) Pastor González, Laura; Geli Fernández-Peñaflor, María Isabel; Universitat de Barcelona. Facultat de Medicina
[eng] CK2 is a constitutively active serine/threonine kinase that consists in a tetramer composed by two catalytic and two regulatory subunits (termed Cka1, Cka2, Ckb1 and Ckb2 in yeast, respectively). Catalytic subunits drive the enzyme activity independently of regulatory subunits, which stabilize the tetramer and serve as a docking platform for the majority of targets. CK2 is vital for cell viability, involved in processes like cell growth, apoptosis or stress responses, yet its dysregulation is associated with diseases, particularly cancer. Increase activity and expression of catalytic subunits along with a downregulation in regulatory ones has been described to promote tumor progression. However, little is known about the regulation of the two CK2 isoforms (tCK2 and mCK2) and the biological consequences of its alteration. Previous research indicates that binding to the phosphoinositide PI(4,5)P2 leads to tCK2 disassembly and inactivation. During the thesis we delve into the mechanism by which PI(4,5)P2 (or other phosphoinositides) regulate the balance between tCK2 and mCK2 and, in turn, understand how they regulate different cellular processes, with particular attention to endocytosis. We were able to define the existence of a lipid binding pocket in Cka1 that resides close to the interface between catalytic and regulatory subunits, in a key region for the constitutive activity of CK2. Modelling the cavity we observed that it can accommodate phosphoinositides such as PI(4,5)P2 and PI4P and inositol polyphosphates as I(1,4,5)P3 or 5-IP7. Structural analysis revealed that Cka1 residues K75-78 bind preferentially to phosphates in position 5 from I(1,4,5)P3 or PI(4,5)P2, while R81, R194, N228 and R230 bind to phosphates located in positions 4 from I(1,4,5)P3, PI(4,5)P2, PI4P or 5 in 5-IP7. The mutation of residues R81, R194, N228, R230 (main) of Cka1 and Cka2 caused their disappearance in the cytosol after fractionations, probably by inducing their aggregation and degradation. We observed that binding to PI4P through R81, R194, N228 and R230 promoted the accumulation of mCK2, as indicated by the native PAGE results from Cka1-main mutant and after depleting the kinases and phosphatases involved in PI4P metabolism. On the contrary, results from Kcs1, the kinase responsible for the pyrophosphate 5-IP7, revealed that the binding of 5-IP7 to these same residues promoted the assembly of tCK2 probably by producing a conformational change in Cka1 β4/β5 loop. Furthermore, we found that the mCK2 isoform, elevated in kcs1∆ and ckb1∆ ckb2∆ mutants, accelerated the dynamics of Myo5, Sla1, Sla2, Ede1, reducing their time anchored to the plasma membrane during endocytosis. These mutants also exhibit higher rates of endocytic events per minute compared to WT cells. Conversely, in the Cka1-main mutant with increased tCK2, the opposite effect is observed, with prolonged lifespans of endocytic complexes and lower endocytic event ratio. Treatment with the CK2 inhibitor TBB, recovered WT dynamics in all of these mutants, indicating that both isoforms were able to phosphorylate endocytic coat components producing different outcomes. Finally, we conducted a phosphoproteome to identify other functional nodes in which mCK2 participated in addition to endocytosis. Results from mCK2 (ckb1∆ ckb2∆) revealed a pool of proteins downregulated, corresponding to exclusive targets of tCK2, but also a pool of upregulated proteins, which may correspond to targets exclusively phosphorylated by mCK2. Gene ontology analysis revealed that both downregulated and upregulated proteins were associated to same processes, being ribosome biogenesis more significant in the upregulated proteins and chromatin organization in the downregulated ones. Given that both groups shared some proteins and processes we hypothesize that, similar to endocytosis, the CK2 isoforms may be controlling the dynamics of protein complexes associated to these processes.
Epigenomic Dynamics Associated with Myeloid Cell Immune Response Remodeling
(Universitat de Barcelona, 2022-06-02) Morante Palacios, Octavio; Ballestar Tarín, Esteban; Universitat de Barcelona. Facultat de Medicina
[eng] Monocytes are highly plastic cells and possess the ability to differentiate into cell types with very different immunological properties. Depending on the external stimuli and the specific immune context, they can give rise to cells that promote immune responses or immune tolerance. These extracellular signals are recognized by specific receptors and integrated by signaling pathways which lead to the participation of transcription factors. Transcriptional remodeling promoted by these transcription factors is generally associated with epigenetic modifications such as histone modifications and DNA methylation. DNA methylation is crucial in the acquisition of immune cells' identity and function. In particular, DNA methylation changes in several human monocytes differentiation models have been previously described. In this thesis, the molecular mechanisms and epigenomic remodeling regulating the development of immunogenic or tolerogenic phenotypes of three clinically relevant monocyte-derived differentiation processes have been studied. Firstly, we investigated the transcriptomic and epigenomic remodeling associated with glucocorticoid-mediated monocyte differentiation to tolerogenic dendritic cells (tolDCs), a cell type that constitutes a potential treatment for various autoimmune diseases. We revealed a major role of MAFB in this process, in synergy with GR. Although both GR and MAFB interact with TET2 and can drive DNA demethylation, the role of MAFB is more extensive, binding to thousands of genomic loci in tolDCs. In this regard, we demonstrated that MAFB knockdown erases the tolerogenic properties of tolDCs and reverts the specific DNA demethylation and gene upregulation. Moreover, in vivo monocyte-derived cells from synovium in rheumatoid arthritis patients treated with glucocorticoids presented an expansion of ‘tolDC-like’ cells with upregulation of MAFB and MAFB target genes. Secondly, we analyzed the effects of vitamin C treatment during monocyte to dendritic cell (DC) differentiation and their subsequent maturation. DNA demethylation has been previously reported to be crucial in that process. Vitamin C is a known cofactor of TET enzymes, which are involved in active demethylation. We outlined extensive vitamin C-mediated demethylation, together with concordant gene expression changes during DC maturation. p65 ( NF-κB) interacts with TET2 and is associated with gene upregulation and DNA demethylation produced by vitamin C treatment. Finally, vitamin C increases TNFβ production and T cell stimulation capabilities of DCs. Thirdly, we integrated the DNA methylation and gene expression remodeling following in vitro exposure of human monocytes to lipopolysaccharide (LPS), a process that generates endotoxin tolerance, a state of hyporesponsiveness to further immune stimuli. In addition, we described phosphorylation of STAT1, STAT3, and STAT5, factors of the JAK2 pathway, after LPS treatment of monocytes. We identified TET2- mediated demethylation and gene upregulation during the first encounter with LPS associated with the JAK2-STAT signaling pathway. In addition, we found that JAK2 inhibition accentuates the tolerant phenotype of monocytes and reduces the expression of tolerized genes. Finally, monocytes from gram-negative septic patients showed lower levels of STAT1 phosphorylation after a second LPS challenge, indicating a reduced JAK2 activity.
Impacto de la respuesta viral sostenida en la recurrencia de la hepatitis C tras el trasplante hepático
(Universitat de Barcelona, 2022-10-24) Matías Mauro, Ezequiel; Crespo Conde, Gonzalo; Navasa, Miquel; Universitat de Barcelona. Facultat de Medicina
[spa] El desarrollo de los nuevos antivirales de acción directa ha permitido lograr la erradicación viral en un porcentaje muy alto de pacientes con infección por VHC, incluso en estadios de fibrosis hepática avanzada y cirrosis descompensada. La erradicación viral y el consiguiente impacto clínico de la misma sobre la historia natural de la enfermedad hepática avanzada, podría dar lugar a un fuerte cambio en la configuración de la LE para TH, no solo en términos de composición etiológica o indicación de TH, sino también en relación a la posibilidad de retirada de LE por mejoría clínica. Por otro lado, tanto el impacto de la erradicación viral en el pre-TH como en el post-TH, podría conllevar a una significativa mejoría en la supervivencia de los pacientes trasplantados con VHC. Así mismo, el hecho de que la erradicación viral conduzca a una sustancial mejoría clínica, de la presión portal, y de marcadores no invasivos de fibrosis del injerto hepático; hace posible elucidar la posibilidad de regresión de la fibrosis hepática, principal vía fisiopatológica involucrada en el desarrollo de las complicaciones hepáticas, y por tanto principal determinante del pronóstico tras la erradicación viral. Sin embargo, a pesar del marcado impacto clínico que acarrea la erradicación viral, a día de hoy se desconocen los principales determinantes asociados a la posibilidad de regresión histológica de la fibrosis, así como también si existe un punto a partir del cual, a pesar de lograr la erradicación viral, la fibrosis establecida resulta irreversible. En igual dirección, el hecho de que la erradicación viral pueda asociarse a una mejoría significativa de la fibrosis, hipertensión portal y marcadores no invasivos de fibrosis en el injerto hepático, hace pensar que la regresión histológica pueda ser significativa, incluso en el subgrupo de pacientes con recidiva del VHC severa, como ser los pacientes con HCF. Finalmente, nuestra tesis encuentra su fundamento en poder establecer dichas hipótesis, a través de un modelo innovador como es el TH, en el cual se conoce con exactitud el tiempo de la infección, factores epidemiológicos, y el estado del injerto al momento de la infección; pudiendo establecer resultados prometedores en la caracterización del proceso de regresión histológica de la fibrosis y su impacto clínico, que podrían ser extrapolables a otras etiologías de enfermedad hepática crónica.
Estudio de las alteraciones genéticas para validar y refinar su impacto pronóstico en leucemia mieloide aguda
(Universitat de Barcelona, 2022-05-19) Bataller Torralba, Alex; Esteve Reyner, Jordi; Universitat de Barcelona. Facultat de Medicina
[spa] La leucemia mieloide aguda (LMA) es la leucemia más frecuente, siendo una neoplasia agresiva con una supervivencia limitada. La LMA presenta mutaciones recurrentes de manera frecuente, y se sabe que estas pueden tener un papel en el pronóstico de la enfermedad. En la presente tesis, se ha validado la clasificación genética European LeukemiaNet (ELN) 2017, que estratifica los pacientes con LMA al diagnóstico en función de ciertas mutaciones. Esta validación se ha realizado en una cohorte de pacientes homogéneamente tratados en el protocolo adaptado al riesgo CETLAM-12. Además de esta validación, se ha identificado un subgrupo de alto riesgo genético con un pronóstico especialmente pobre. Por otro lado, dentro del grupo de riesgo favorable de la ELN 2017, el seguimiento de los pacientes mediante técnicas de enfermedad medible residual (EMR) ha demostrado su impacto pronóstico. En este sentido, se ha evaluado la implementación de una intervención precoz, anticipando el tratamiento en pacientes con recaída molecular. Se ha demostrado que esta intervención precoz ha comportado un beneficio en la supervivencia de estos pacientes con LMA.
Características clínicas e indicadores pronósticos de reingreso y mortalidad de una cohorte de pacientes que ingresan por primera vez por insuficiencia cardíaca en un hospital universitario de tercer nivel: un estudio observacional de 5 años
(Universitat de Barcelona, 2021-12-14) Masip Masip, Joan; Formiga Pérez, Francesc; Corbella, Xavier; Universitat de Barcelona. Facultat de Medicina
[spa] La Insuficiencia cardiaca es una condición de elevada prevalencia cuya frecuencia aumenta con la edad y tiene un impacto notable en el gasto sanitario. Los primeros meses tras el alta de la primera hospitalización son críticos para el pronóstico de los pacientes. MATERIAL Y MÉTODOS: Se realizó un seguimiento de un año a todos los pacientes tras un primer ingreso debido a IC en el periodo 2.010-2.014 en el Hospital Universitari de Bellvitge. Las fuentes de datos fueron el Conjunto Mínimo Básico de Datos al Alta Hospitalaria (CMBDAH), registros de Atención Primaria, Actividad Hospitalaria y Registro Central de Asegurados (para comprobar el estatus vital al final del seguimiento). El estudio se dividió en tres artículos: 1/Validación de un modelo predictivo de mortalidad y readmisión (RR Score). 2/Estudio descriptivo de la primera hospitalización por IC, donde se determinó la tasa de mortalidad hospitalaria y perfil del paciente. 3/Estudio del pronóstico a 30 días tras el alta del primer ingreso en cuanto a muerte, readmisión no programada y factores asociados. RESULTADOS: Se registraron 132.989 episodios. La prevalencia de IC fue del 12,2% (16.273 episodios), supuso la causa de ingreso en 6.816, el 5,1% del total y 3.868 (el 56,7%) correspondían a un primer ingreso. La edad media fue de 75,8 años (SD=12) y el 53,8% eran mujeres. Las comorbilidades más frecuentes fueron HTA (63,1%), fibrilación auricular (36,8%), dislipemia (35,8%) y Diabetes (33,7%). El ingreso urgente fue la vía de entrada más común (3.551 pacientes, 91,8%). La tasa de mortalidad hospitalaria fue del 5,8% (226 pacientes) sin diferencias significativas entre hombres y mujeres. En el análisis multivariante, la estancia hospitalaria prolongada (OR=1,90, p<0,001), la IRC (OR=1,44, p=0,02), la edad (OR = 5,57 en los mayores de 85 años) y la admisión en la unidad de críticos (OR=4,97, p<0,001) eran factores predictivos de una mayor mortalidad. En los 3.642 pacientes dados de alta con vida, la distribución por sexo y edad no difería de la muestra inicial. La tasa de readmisión por IC a 30 días fue del 9,3. Ser atendido en urgencias tras el alta fue predictivo de un reingreso (OR 6,97). La tasa de mortalidad a los 30 días fue del 5.6%, con una media de edad de 81 años, (SD=12). La atención en urgencias tras el alta (OR 2,31) y la edad mayor de 70 años (OR 2,78) eran los principales predictores de mortalidad. El modelo predictivo RRScore mostró estadísticos C de 0,649 para readmisión por cualquier causa a 30 días y de. 0,621 para las readmisiones a 90 días. La IC fue el diagnóstico principal de ingreso en el 5,1% de los episodios registrados. De estos, el 56,7% correspondían a un primer episodio de descompensación. CONCLUSIONES: Dos patrones clínicos motivaron un primer ingreso: a)mujeres de edad avanzada con alta prevalencia de comorbilidades, ingresos cortos y elevada mortalidad hospitalaria b) hombres menores de 65 años, con ingreso programado para cirugía de recambio valvular, propio de un centro de alta especialización. La mortalidad del primer ingreso fue del 5,8% y las tasas de readmisión y mortalidad en los 30 días posteriores fueron mayores que las publicadas en otros países de la UE. Los factores asociados a muerte y/o readmisión en un periodo de 30 días tras el alta de una primera hospitalización por IC fueron la edad, comorbilidades y necesidad de atención urgente previa o posterior al primer ingreso. El Readmission Risk Score mostró una capacidad modesta de identificar los pacientes a riesgo de readmisión y no debería recomendarse su uso en IC de reciente aparición. Los resultados de la presente Tesis han servido como herramienta básica el diseño e implementación en 2016 de la Unidad Multidisciplinar de Insuficiencia Cardíaca Comunitaria (UMICO), encargada de mejorar la calidad, seguridad y continuidad asistencial de la atención a los pacientes con IC.
Hipertensión portal: Avances en su diagnóstico e impacto en el riesgo quirúrgico
(Universitat de Barcelona, 2021-04-08) Ferrusquía Acosta, José Alberto; Hernández Gea, Virginia; García Pagán, Juan Carlos; Universitat de Barcelona. Facultat de Medicina
[spa] La hipertensión portal es un síndrome clínico que consiste en el aumento patológico y sostenido de la presión en el territorio venoso portal. Su relevancia se ve determinada por las graves complicaciones que produce, las cuales condicionan una elevada morbilidad y mortalidad en pacientes con enfermedades crónicas del hígado. El gradiente de presión venosa hepática, la diferencia entre la presión suprahepática enclavada y la presión suprahepática libre, es la técnica de elección para evaluar la hipertensión portal en pacientes con cirrosis, ya que proporciona información pronóstica relevante relacionada con la supervivencia y el riesgo de descompensación. No obstante, su valor pronóstico depende de la existencia de una buena correlación entre la presión suprahepática enclavada y la presión portal. Por desgracia, los estudios que han demostrado que la presión suprahepática enclavada es capaz de estimar con precisión la presión portal no han incluido a pacientes con enfermedad hepática grasa no alcohólica, la cual se ha posicionado como la causa más frecuente de hepatopatía crónica en nuestro medio. Por otra parte, los avances continuos en las técnicas quirúrgicas y el manejo médico han dado lugar a un aumento en el número de pacientes con hipertensión portal que son derivados para una evaluación prequirúrgica. Por lo tanto, es fundamental comprender los riesgos y los beneficios de la realización de procedimientos quirúrgicos en pacientes con enfermedades crónicas del hígado. La mayoría del conocimiento actual sobre el riesgo quirúrgico en pacientes con hipertensión portal proviene de estudios realizados en pacientes con cirrosis. Sin embargo, la evidencia relacionada con el pronóstico postquirúrgico en pacientes con hipertensión portal idiopática es escasa y se limita a cirugías cada vez menos indicadas como la esplenectomía o la creación quirúrgica de una derivación portosistémica. Los trabajos incluidos en la presente tesis pretenden evaluar, por un lado, la correlación entre la presión suprahepática enclavada y la presión portal en pacientes con enfermedad hepática grasa no alcohólica, y por otro, el pronóstico postquirúrgico de los pacientes con hipertensión portal idiopática que son sometidos a una cirugía abdominal.
Asthma and obesity. Glucocorticoid sensitivity before and after bariatric surgery. Effect of vitamin D
(Universitat de Barcelona, 2022-12-21) Bantulà, Marina; Arismendi Núñez, Ebymar; Roca i Ferrer, Jordi; Universitat de Barcelona. Facultat de Medicina
[eng] This thesis has been designed to characterize the low-grade chronic inflammation present in obesity in patients suffering from asthma, and to describe some of the mechanisms involved in the glucocorticoid hyporesponsiveness seen in clinical practice. Moreover, the effects of weight loss through bariatric surgery on the inflammatory status and on glucocorticoid response of the asthma-obesity association have been defined. Finally, the vitamin D effect on cell proliferation and on glucocorticoid sensitivity has been discussed. It seems quite well demonstrated that obesity is associated with asthma, however, the mechanisms that link the two processes are surely very complex. The inflammatory process underlying both diseases could be one of the potential connections between them. The excessive accumulation of adipose tissue in subjects with obesity results in an increased production of pro-inflammatory cytokines (IL-1β, IL-6, IL- 18, TNF-α, MCP-1) and adipokines such as leptin, and a reduction in the release of adiponectin, with anti-inflammatory properties. This low-grade chronic systemic inflammation present in obesity contributes towards increasing the systemic and/or airway inflammatory process underlying asthma. Inhaled corticosteroids (ICS) are considered the cornerstone of controller therapy for asthmatic patients. ICS have the ability to reduce airway inflammation, airway obstruction, airway hyperresponsiveness, and asthma symptoms. However, asthmatic patients with obesity are more likely to have a poor response to glucocorticoids: they have reduced odds of achieving asthma control, higher risk of asthma hospitalizations, and lower quality of life compared with asthmatics with a normal body mass index. Detrimental effects of obesity on lung function and additive or synergistic effects of obese systemic inflammation on airways inflammation, have been proposed as potential mechanisms to explain glucocorticoid hyporesponsiveness in asthmatic patients with obesity. Moreover, a defective induction of anti-inflammatory genes by glucocorticoids, such as mitogen-activated protein kinase phosphatase-1 (MKP-1), has been described as another possible mechanism of the reduced response to glucocorticoids seen in asthmatic patients with obesity. Moreover, bariatric surgery is considered the most effective and sustained long-term treatment of severe obesity. Weight reduction significantly improves systemic and adipose tissue inflammatory activity levels. However, the effect of weight loss on specific molecules potentially involved in the inflammatory process in obesity and in obesity-related asthma remains to be examined. Several studies have shown an improvement in asthma control, medication use, hospitalization rate, and lung function after weight loss via bariatric surgery. However, no studies have assessed glucocorticoid sensitivity before and after bariatric surgery. Finally, vitamin D is a hormone with pleiotropic effects and numerous regulatory mechanisms beyond bone health. Evidence from observational studies suggests that obesity is associated with vitamin D deficiency. Furthermore, low serum vitamin D levels have been associated with asthma exacerbations. Other reports described an association between vitamin D deficiency and the risk of glucocorticoid resistance. There is also in vitro evidence for vitamin D increasing glucocorticoid sensitivity. In peripheral blood mononuclear cells from steroid resistant asthmatic patients, the active form of vitamin D (1,25- dihydroxyvitamin D) increases the expression of MKP-1, thus enhancing glucocorticoid inhibition of cell proliferation.
Pre-clinical development of chimeric virus-like particles based HPV: HIV vaccines by using mammalian cell expression system: limitations and challenges
(Universitat de Barcelona, 2022-11-08) Chen, Chun-Wei; Joseph Munné, Joan; Universitat de Barcelona. Facultat de Medicina
[eng] HPV and HIV-1 are important public health issues in some developing and industrialized countries, but an effective chimeric HPV:HIV preventive vaccine is still unachievable. We aimed to establish an alternative mammalian (293F) cell expression system combining with chromatographic purification methods to reach an appreciable expression level, purity and recovery rate of chimeric HPV:HIV VLPs. In this study, the chimeric HPV:HIV (L1:P18I10 and L1:T20) immunogens were designed and produced by using 293F expression system. The HPV:HIV VLPs were subsequently purified by a 3-step chromatographic method, including cation (CEC), size exclusion (SEC) and heparin affinity (H-AC) chromatography. Then, the in vitro stability, in vitro self assembly and morphology of purified HPV:HIV VLPs were confirmed by non-reducing SDS-PAGE, molecular mass assay, transmission electron microscopy (TEM) respectively. The sequential and conformational P18I10 and T20 peptides presented on chimeric HPV:HIV VLPs were further characterized by HIV-1 anti-V3 and anti-2F5 monoclonal antibodies in vitro by using Western blot and indirect ELISA. Finally, the immunogenicity of HPV:HIV VLPs were assessed in BALB/c mice model. Because the development and manufacturing of an immunogenic HPV:HIV vaccine is still unachievable, this study provided a baseline strategy that may be worthy to support the global efforts to develop novel chimeric VLP-based vaccines for controlling HPV and HIV-1 infections
ldentification of pTINCR: a new ubiquitin-like protein that promotes epithelial differentiation and suppresses tumor growth
(Universitat de Barcelona, 2022-10-11) Boix Sánchez, Olga; Abad Méndez, María; Felip Font, Enriqueta; Universitat de Barcelona. Facultat de Medicina
[eng] Recent advances in computational analyses, peptidomics and ribosome profiling have revealed that many regions previously annotated as non-coding are in fact translated into a myriad of bioactive proteins, largely overlooked until now. These small proteins, shorter than 100 amino acids, are called microproteins, micropeptides or SEPs (from sORF-encoded peptides). To date, only a subset of them have been functionally characterized, and they have been shown to play essential functions regulating a plethora of fundamental processes such as DNA repair, RNA splicing, or cell metabolism. Importantly, mounting evidence suggest that microproteins also have a role in response to stress, such as oxidative stress, viral and bacterial infection, tissue injury and even tumor initiation. The acquisition of cell identity by cell differentiation is essential for proper tissue function. In epithelial tissues, cell polarity plays a pivotal role during tissue morphogenesis and in the maintenance of the specialized functions and differentiated status of epithelial cells. On the other hand, loss of epithelial cell identity is nowadays considered an essential initial step in tumorigenesis, and epithelial cell polarity has been positioned as a key tumor suppressive mechanism in epithelial cancers. In this doctoral thesis, we aimed to find and characterize novel microproteins with an important role in cell identity and cancer. We have identified pTINCR, an 87-amino acid microprotein encoded by TINCR, a gene misannotated as a long non-coding RNA. pTINCR is an evolutionary conserved microprotein expressed in skin and several epithelial tissues. By gain- and loss-of-function studies, we have demonstrated that pTINCR is a key regulator of epithelial cell differentiation in vitro and promotes epidermal differentiation in vivo. Additionally, pTINCR is upregulated upon cellular damage by the tumor suppressor p53 and it is required for the DNA damage-induced differentiation response. Consistent with its tumor suppressive role, pTINCR expression is lost in human cutaneous squamous cell carcinomas (cSCCs), and its overexpression reduces malignancy in cSCC patient-derived xenografts (PDXs). Moreover, the expression of TINCR correlates with better prognosis in several epithelial cancers. At the molecular level, pTINCR is a novel ubiquitin-like protein (UBL) which can bind to SUMO through its SUMO interacting motif (SIM) and regulate SUMO conjugation. Importantly, we have identified two pTINCR binding partners, NONO and CDC42, and showed that pTINCR enhances their SUMOylation. NONO is a nuclear protein involved in transcriptional regulation, pre-mRNA splicing and nuclear retention of defective RNAs. We have shown that pTINCR triggers a specific splicing program related to epithelial cells, probably through its interaction with NONO. On the other hand, CDC42 is a Rho-GTPase critical for actin cytoskeleton remodeling and the establishment of cell polarity. We demonstrate that pTINCR binds to CDC42 and promotes its SUMOylation and activation, triggering an epithelial pro-differentiation cascade. In summary, we have identified pTINCR as a novel UBL that regulates epithelial differentiation and has tumor suppressor activity. Our results further expand our knowledge on the yet unexplored field of microproteins and suggest that the proteome hidden in previously assumed non-coding RNAs can indeed be a source of new regulators of cell identity relevant for cancer.
Production, purification and characterization of virus-like particles from chimeric HPV-HIV L1P18 protein expressed in Pichia pastoris: Lessons learned
(Universitat de Barcelona, 2021-10-29) Eto, Yoshiki; Joseph Munné, Joan; Aligué i Alemany, Rosa Maria; Universitat de Barcelona. Facultat de Medicina
[eng] In this thesis, two recombinant Pichia pastoris strains producing chimeric HPV-HIV L1P18 protein were constructed. One intracellularly produces the chimeric protein while the other secrets it. For the former, several purification methods were systematically tested and the purification process was optimized. After cell lysis, virus like particles (VLPs) from L1P18 protein were purified by a combination of ammonium sulfate precipitation, size exclusion chromatography, ultracentrifugation and ultrafiltration. At the end of the purification process, the chimeric VLPs were recovered with 96% purity and 9.2% overall yield, and the morphology of VLPs were confirmed by transmission electron microscopy. This work contributes towards the development an alternative platform for production of a bivalent vaccine against HPV and HIV in P. pastoris.
The role of RANK pathway in the intestinal epithelium, breast cancer and tumor immune modulation
(Universitat de Barcelona, 2021-10-25) Gómez Aleza, Clara; González Suárez, Eva; Universitat de Barcelona. Facultat de Medicina
[spa] La vía de RANK juega un papel crucial en remodelación ósea, con un anticuerpo monoclonal (denosumab) contra el ligando de la vía de señalización (RANKL), actualmente usado en clínica contra la osteoporosis y efectos adversos derivados de metástasis óseas. RANK es también crucial en el desarrollo de la glándula mamaria y media procesos de tumorogénesis en el epitelio mamario. También se ha descrito su papel en el sistema inmune, donde es necesario para la formación de nódulos linfáticos, la maduración de células dendríticas y el desarrollo de linfocitos T en el timo. Dada la importancia de la vía de RANK en estos diversos contextos, el objetivo de esta tesis es investigar el papel de esta vía de señalización en la comunicación entre células epiteliales y el sistema inmune en contextos tumorales. Usando modelos murinos de tumorogénesis causada por la expresión de un oncogén (PyMT), identificamos que la vía de RANK establece un entorno inmunosupresor en los transplantes de tumores, mediada por un mayor reclutamiento de neutrófilos y una menor infiltración de células T CD8+ anti-tumorales. En ausencia de expresión de RANK, los tumores retrasan su crecimiento, pero finalmente consiguen evadir el sistema inmune con una mayor infiltración de células T reguladoras y la expresión de "immune checkpoints" (CTLA-4, PD-L1 y PD-1) en el microentorno tumoral. Por tanto, los tumores en los que se inhibe la señalización a través de RANK por medio de depleción genética o inhibición farmacológica del ligando, presentan una mayor respuesta a las combinaciones de anticuerpos contra los "immune checkpoints". Relevantemente, estas observaciones se confirman en pacientes de cáncer de mama temprano tratadas con denosumab, donde la inhibición de RANKL causa una mayor infiltración de células T en el tumor. En esta tesis doctoral, también se explora el papel de la vía de RANK en modelos de tumorogénesis derivada de inflamación crónica en el colon. En este modelo, la pérdida de RANK no supone un cambio en la agresividad de los tumores o su crecimiento, pero se observó una correlación inversa entre la expresión de RANK y marcadores de activación de la vía de WNT, la cual está asociada con la mayoría de tumores de colon. Por tanto, en esta tesis se ha asociado la vía de RANK con el establecimiento de un ambiente inmunosupresor en el cáncer de mama, con implicaciones para el futuro de la inmunoterapia en este tipo tumoral clásicamente asociado a una menor respuesta a este tipo de fármacos. Así mismo, también se ha establecido una correlación inversa entre marcadores de proliferación/actividad de célula madre en intestino y colon con la expresión de la vía de RANK, sugiriendo un papel distinto para esta vía de señalización en el epitelio intestinal comparado con su papel descrito en el epitelio mamario.
The role of cyclin-dependent kinase 5 (Cdk5) in colorectal cancer
(Universitat de Barcelona, 2021-07-20) Kjaergaard Bystrup, Sara; Martínez Balibrea, Eva; Universitat de Barcelona. Facultat de Medicina
[cat] El càncer colorectal (CCR) és el tercer càncer més freqüent en homes i el segon en dones a nivell mundial. Tot i que les taxes de supervivència a 5 anys en els primers estadis superen el 90%, la situació canvia dràsticament en pacients metastàtics. S’han vist pocs avenços en el tractament del CCR en les darreres dècades, el qual encara consisteix en diferents règims de quimioteràpia, amb la possibilitat de ser combinats amb una de les poques teràpies dirigides que són efectives. Per tant, es necessita trobar noves dianes terapèutiques, juntament amb biomarcadors pronòstics i predictius per optimitzar l’eficiència dels tractaments actuals i futurs. La cinasa dependent de la ciclina 5 (Cdk5) és més coneguda per les seves funcions en el sistema nerviós, però en l'última dècada s'ha demostrat que també està involucrada en molts processos propis del càncer. En aquest estudi, hem investigat l’expressió de Cdk5 en línies cel·lulars de CCR i en un gran nombre de mostres tumorals per tal d’avaluar la seva rellevància en aquesta patogènesi. Vam trobar que Cdk5 s’expressa i està molt activa en les línies cel·lulars de CCR i tot i que no afecta la proliferació, el seu silenciament gènic disminueix la invasió i la migració cel·lular, particularment en les línies cel·lulars amb mutacions activadores per sota del receptor del factor de creixement epitelial. Els anàlisis d’enriquiment funcional i els efectes transcripcionals com a conseqüència de l’eliminació del gen CDK5, també van confirmar la participació d’aquesta proteïna en la motilitat del càncer. En els teixits tumorals, Cdk5 es troba sobre-expressada en comparació amb els teixits normals a causa d’un guany en el nombre de còpies. En pacients amb malaltia localitzada, vam trobar que els nivells alts de Cdk5 es correlacionen amb un pitjor pronòstic, especialment en pacients que tenen mutacions en KRAS. En el context metastàtic, Cdk5 prediu la resposta en pacients tractats amb oxaliplatí, essent aquest fet corroborat per estudis in vitro amb línies cel·lulars resistents a l’oxaliplatí i no donant-se en el cas de l’irinotecan. En explorar els nivells de Cdk5 en els subtipus moleculars consensuats, vam trobar els nivells més baixos en el subtipus 1, on uns nivells elevats de Cdk5 es va tornar a associar amb un pitjor pronòstic, tot i que els estudis in vitro, in vivo i ex vivo no indiquen un paper per a la Cdk5 en infiltració immunitària o resposta al bloqueig del punt de control immunitari en CCR. Els resultats d’aquesta tesi doctoral confirmen la rellevància de Cdk5 en CCR, i tot i que es necessiten estudis addicionals, suggerim que la Cdk5 es podria utilitzar com a biomarcador per a la teràpia adjuvant en CCR en estadis inicials o per predir la resposta a quimioteràpies. Alternativament, la inhibició de la Cdk5 es podria utilitzar en la adjuvancia per evitar la recaiguda de la malaltia o en un entorn metastàtic en combinació amb oxaliplatí.
Ancestral roles of IĸB proteins in Caenorhabditis elegans
(Universitat de Barcelona, 2021-07-09) Ahumada Brena, David Ricardo; Cerón Madrigal, Julián
[eng] Mammalian Inhibitor-of-kappa-B (IκBs) proteins exert their main function as negative regulators of NF-κB, a central signaling pathway controlling immunity and inflammation. An alternative chromatin role for IκBs has been shown to affect stemness and cell differentiation. However, the involvement of NF-κB in this function has not been excluded. NFKI-1 and IKB1 are IκB homologs in aenorhabditis elegans, which lacks NF-κB nuclear effectors. We found that nfki-1 and ikb-1 mutants display developmental defects that phenocopy mutations in Polycomb and UTX-1 histone demethylase, suggesting a role for C. elegans IκBs in chromatin regulation. Further supporting this possibility (i) we detected NFKI-1 in the nucleus of cells; (ii) NFKI-1 and IKB-1 bind to histones and Polycomb proteins, (iii) and associate with chromatin in vivo, and (iv) mutations in nfki-1 and ikb-1 alter chromatin marks. Based on these results, we propose that ancestral IκB proteins modulate chromatin marks with an impact on development.
Dissecting genetic regulatory mechanisms in human pancreatic islets to gain insights into type 2 diabetes pathophysiology
(Universitat de Barcelona, 2021-05-05) Atla, Goutham; Ferrer Marrades, Jorge; Bonàs Guarch, Sílvia; Universitat de Barcelona. Facultat de Medicina
[eng] Diabetes mellitus is a heterogeneous group of metabolic diseases characterized by impaired blood glucose homeostasis that affects more than 415 million people worldwide and is a leading cause of mortality. The most prevalent form of diabetes is Type 2 Diabetes (T2D) that accounts for 90% of diabetes cases. An interplay of environmental and genetic risk factors contributes to etiology of T2D via a progressive loss of pancreatic beta cell function coupled with insulin resistance. Genome Wide Association Studies (GWAS) identified more than 400 independent genetic loci associated with T2D risk, although the molecular mechanisms underlying these genetic signals remain poorly understood. A comprehensive understanding of gene regulation in human pancreatic islets and identifying the role of T2D risk variants on different components of gene regulation will enlighten our insights into T2D etiology. In this work, we performed an in-depth characterization of human pancreatic islets transcriptional regulatory elements, attaining a greater granularity at transcriptional enhancers. We further identified glucose responsive enhancers which regulate glucose-dependent gene expression programs via three-dimensional chromatin interactions. This allowed us to gain insights into human islet transcriptional gene regulation and how glucose, a primary physiological stimulant of pancreatic islets, modulates human islet genome function. We also generated comprehensive transcriptome annotations in human islets using short- and long-read sequencing data along with accurate maps of transcriptional start sites. This revealed islet-specific promoters, transcript isoforms and novel coding sequences. This underscored the importance of generating transcript models in disease relevant tissue to progress in the understanding of gene regulation. Finally, these parallel efforts allowed us to create pioneer maps of genetic effects on human alternative splicing that revealed for the first time the noteworthy contribution of human islet mRNA splicing to T2D pathophysiology. These results have thus the potential to blossom in the discovery of novel T2D drug targets.
Identity and functions of dendritic cell subsets in ischaemia-induced neuroinflammation
(Universitat de Barcelona, 2021-02-15) Gallizioli, Mattia; Planas Obradors, Anna Maria; Miró Mur, Francesc; Universitat de Barcelona. Facultat de Medicina
[eng] Cerebral ischaemia induces several inflammatory processes in the brain. Among them, the infiltration of immune cells is a hallmark of the pathology. Dendritic cells (DCs) are usually present in low numbers in the meninges and the choroid plexus, but rarely in the parenchyma. Upon ischaemia, the number of DCs increases, and the cells infiltrate the brain tissue, where they carry out different functions. In an experimental murine model of stroke, we set out to investigate the infiltration of several subsets of DCs to the brain and their functional role. Early after stroke, we show a rapid and significant influx of DCs, especially of conventional type 2 DCs (cDC2), which are the most abundant subset at all time points analysed. Twenty-four hours after stroke, these cells were the major source of IL-23, which was able to stimulate its receptor on γδ T cells, inducing their production of IL-17. In turn, IL-17 is responsible for the stimulation of the production of Cxcl1 by astrocytes, ultimately leading to the infiltration of neutrophils to the ischaemic brain and to the exacerbation of the tissue damage. We demonstrate that the interruption of the IL-23/IL-17 axis decreases the infarct size and improves the neurological outcome of stroke in mice, suggesting that cDC2 may play a detrimental role in the early phase of the immune response to stroke. The analysis of the infiltration of DCs to the brain in inflammatory conditions has historically been difficult for the absence of univocal markers and for the similarity of their phenotype with other brain cells, especially microglia. The knowledge about the origin, phenotype and functions of brain DCs is therefore underdeveloped. One of the most commonly used markers for the study of DCs is CD11c, which is also expressed by a subset of microglia. The population of CD11c+ cells present in the brain increases after stroke, and we show that CD11c+ cells include proliferating microglia and infiltrating DCs. Despite their similarities, we demonstrate by RNA-Seq analysis that these two cell types exhibit a differential transcriptional profile, with interesting peculiarities in pattern recognition receptor and chemokine receptor expression. DCs extracted from the ischaemic brain outclass microglia in antigen presentation capacity, indicating a functional specialisation. We show that microglia are responsible for the production of chemokines that attract DCs to the brain, especially conventional type 1 DCs (cDC1). This specific subpopulation of DCs appears to have beneficial functions, reducing the infarct size and improving the functional outcome of ischaemic stroke. Altogether, the studies presented in this thesis shed light on the features discriminating DCs from microglia and uncover previously unknown roles of diverse subpopulations of infiltrating DCs in the outcome of ischaemic stroke.
Combined Therapies for Neuroblastoma Based on the Activation of the Calcium- Sensing Receptor
(Universitat de Barcelona, 2021-01-15) Gonçalves Alves, Eliana Carolina; Lavarino, Cinzia; Mateo Lozano, Silvia; Universitat de Barcelona. Facultat de Medicina
[eng] Neuroblastoma (NB) is the most common extracranial solid childhood tumour. Its clinical and histological manifestations range from benign tumours that spontaneously regress to highly aggressive metastatic tumours (Matthay et al -2016). In spite of the advances in treatment and the multidisciplinary approaches, almost half of high-risk NB patients do not survive (Whittle et al – 2017). The Calcium-sensing receptor (CaSR) is a G-protein coupled receptor (GPCR) that was found to be expressed in good prognosis NB tumour (de Torres et al – 2009). Activation of this receptor using cinacalcet (CIN), a positive allosteric modulator of CaSR, in a xenograft NB animal model, reduced tumour growth. In addition, it was described that in NB cell lines with an overexpression of CaSR, CIN induced ER-stress mediated apoptosis (Rodríguez-Hernández et al – 2016). However, efficacy of CIN in the treatment of NB is limited by the low expression of CaSR in high-risk NB. Furthermore, CIN acts mainly in the CaSR present in the parathyroid glands, inducing hypocalcemia. This work addresses these two limitations of using CIN in the treatment of NB patients. We demonstrate that the active compound of vitamin D, 1,25-dihydroxyde vitamin D (1,25-D3) and the retinoids all-trans-retinoic acid (ATRA) and 13-cis-retinoic acid (13-cis-RA) increase the expression of CaSR in two NB cell lines. Unfortunately, in vitro anti-tumorigenic capacities of CIN are not increased by its combination with 1,25-D3. Moreover, we show that the strong in vitro and in vivo anti-tumorigenic capacities of retinoids are unaltered by its combination with CIN. On the other hand, we identify another positive allosteric modulator of CaSR, AC-265347, with NB anti-tumorigenic properties which does not induce hypocalcemia in mouse animal models. In these models each calcimimetic induce a differential protein and gene expression pattern, suggesting a different mechanism of action in the inhibition of tumour growth. Additionally, in vitro studies show that CIN and AC-265347 induce different stages of differentiation in NB cell lines. Altogether, our data shows that the combination of CIN with different drugs that induce an increase in the expression levels of CaSR do not potentiate the anti-tumorigenic effect of CIN. More importantly, this work identifies a new calcimimetic that shows a potential neuroblastoma specific effect, AC-265347 inhibits NB tumour growth while maintaining plasma calcium levels. Our results strongly suggest that AC-265347 may be and effective therapeutic agent against NB, alone or in combination with other potentially synergistic treatments.
Role of CD44 in clear cell renal cell carcinoma invasiveness after antiangiogenic treatment
(Universitat de Barcelona, 2020-12-09) Aparicio García, María; Casanovas i Casanovas, Oriol; Universitat de Barcelona. Facultat de Medicina
[eng] The arrival of antiangiogenic therapies represented a huge advance in the treatment of different tumors, such as renal carcinoma. However, despite initial good results, some tumors were able to develop different mechanisms to adapt to the new conditions and continue growing. Thus, resistance to the antiangiogenic drugs appeared. Moreover, preclinical studies showed that some tumors increased their invasive and metastatic capacity after the therapy. Different murine orthoxenografts models, derived from patients with renal carcinoma, were developed to characterize the response to the antiangiogenic treatment. Both drugs used, DC101 and Bevacizumab, produced a reduction of the tumor growth and its volume, as well as a reduction of the number of vessels. However, when tumor invasion was evaluated, it could be observed that not all tumor models responded equally. While some tumors showed a higher metastatic capacity and an increase in invasiveness and aggressiveness after the treatment, others did not show any alteration of their characteristics. To study the mechanisms involved in the increase of tumor malignization, different assays comparing the gene expression of non-invasive and pro-invasive tumors after the antiangiogenic treatment were developed. These analyses generated a list of candidate genes that could be responsible for the invasive process, with CD44 among them. In this thesis, we have studied the role of CD44 in the tumor invasion and migration processes using in vitro and in vivo models of renal carcinoma. We have explored the signaling pathway that could be induced due to the CD44 activation. The Src signaling route was possibly shown to be involved in the invasive process of cancer cells. The mechanism responsible for CD44 activation has also been investigated. Between different candidates, Serglycin appeared as a possible ligand and inducer of CD44. Thus, this thesis opens different study ways to determine the molecules responsible for tumor invasion after antiangiogenic treatment and develop new different strategies to overcome the resistance.
Molecular mechanisms of resistance to therapy in glioblastoma
(Universitat de Barcelona, 2020-10-30) Pedrosa, Leire; Iglesia Zaragoza, Núria de la; Universitat de Barcelona. Facultat de Medicina
[cat] El glioblastoma (GBM) és el tumor primari cerebral més freqüent i més agressiu. Els pacients amb GBM tenen una supervivència mitjana de 15 mesos. Una dels principals dificultats que presenten els GBMs per ser tractats és la seva elevada heterogeneïtat inter-tumoral (entre pacients) i intra-tumoral (dins d’un mateix tumor). El GBMs es poden classificar en quatre subtipus segons el seu perfil epigenètic i expressió gènica: G-CIMP+ i tres G-CIMP- anomenats proneurals (PN), clàssics (CL) i mesenquimal (MES). S’ha observat que aquests subtipus moleculars no són estàtics i poden presentar transicions d’un subtipus a un altre. A més, dins d’un mateix tumor hi podem trobar diferents tipus cel·lulars, com les cèl·lules mare iniciadores de gliomes (GICs) que tenen capacitat d’auto-renovar-se i generar el tumor de nou. A més, les GICs també es poden classificar en els subtipus moleculars i poden fer transicions d’un subtipus a un altre. Aquesta tesis doctoral consta de dos projectes que es centren en identificar possibles mecanismes moleculars que confereixen resistència a les teràpies actuals als GBMs. En el primer projecte hem identificat dos long non-coding RNAs (lncRNAs) que podrien regular la capacitat d’auto-renovació de les GICs, la qual permet generar el tumor de nou, fent recidivar el GBM. En el segon projecte hem corroborat la funció de GPR56 en la diferenciació de les GICs al subtipus MES, procés correlacionat amb la resistència a les teràpies en GBMs. Pel projecte dels lncRNAs, vam fer RNA-seq de mostres de teixit tumoral de GBM. Les mostres es van classificar segons el subtipus de GBM i es va realitzar un network anàlisis ponderat de co-expressió gènica (WGCNA) per tal de seleccionar els lncRNAs candidats. Els tres lncRNAs candidats van ser PAUPAR, PROCAR i LINC00941, els quals es co-expressaven amb algun dels gens principals dels subtipus de GBM i tenien un elevat valor de predicció d’importància dins del network. Per estudiar-los uncionalment, vam generar GICs amb knockdowns dels lncRNAs candicats, mitjançant short hairpin RNAs (shRNAs). Amb aquest mètode, vam aconseguir noquejar l’expressió PAUPAR en GICs clàssiques i el LINC00941 en les GICs mesenquimals. Finalment, vam estudiar si la capacitat d’auto-renovació de les GICs estava afectada per la pèrdua d’expressió dels lncRNA i si hi havia alguna transició entre els diferents subtipus de GBM. Les shlncRNA-GICs formen menys neurosferes que les control, indicant que les GICs amb menys expressió d’aquests lncRNAS tenen disminuïda la capacitat d’auto-renovació. A l’estudiar l’expressió de varis gens dels subtipus de GBM amb RT-qPCR i citometria de flux, no es van observar transicions entre els subtipus. Per tant, PAUPAR i LINC00941 podrien estar mantenint la capacitat d’auto-renovació de les GICs afavorint la recurrència dels pacients després dels tractaments sense intervenir en les transicions dels subtipus de GBM. Per altra banda, per estudiar la funció de la proteïna de membrana GPR56 en les GICs, vam generar GICs proneurals amb knockout per GPR56, mitjançant CRISPR, i GICs clàssiques amb knockdown de GPR56, mitjançant shRNAs. Un cop generades les línies amb menys expressió de GPR56, vam estudiar diferents marcadors MES per citometria de flux i per RT-qPCR, observant un augment dels marcadors MES en les KO-GPR56-PN-GICs i en les shGPR56-CL-GICs. Aquests resultats suggereixen que GPR56 està inhibint la diferenciació de les GICs PN i CL al subtipus MES. Per tant, GPR56 podria ser important en la inhibició del guany de resistència a la teràpia. Per tant, podem dir que hem identificat dos molècules que podrien estar involucrades en mecanismes de resistència a les teràpies. PAUPAR i LINC00941 podrien estar involucrats en la recurrència dels pacients mantenint la capacitat d’auto-renovació de les GICs i GPR56 inhibint la diferenciació cap al subgrup MES, la qual augmenta la resistència a les teràpies en GBM.
Estudio clínico de la fiebre tifoidea
(Universitat de Barcelona, 1915-01-27) Montrós Perelló, Juan J.; Universitat de Barcelona. Facultat de Medicina
[spa] A la hora de escoger un tema para la Memoria de doctorado pensamos en seguida en el que tenemos el honor de proponer a este Iltre. Tribunal, a saber: el estudio clínico de la fiebre tifoidea. Nos limitaremos al estudio clínico para confirmar la importancia capital de la clínica sobre todo lo demás, para dejar ver que el tratamiento será tanto más eficaz cuanto más se funde en la fisiopatología clínica, y para demostrar que el laboratorio y los libros de texto, auxilios preciosísimos del médico, son inútiles por completo cuando no se colocan y estudian junto a las camas de un hospital. Dicho esto, vamos a entrar en materia, para lo cual, ante todo, hemos de dividir nuestro trabajo en capítulos, que serán ocho, correspondientes a: Etiología y patogenia; fisiología patológica; sintomatología; evolución, diagnóstico; tratamiento y profilaxia, todo ello desde el punto de vista clínico. Y así, vamos a limitarnos a lo necesario, a lo que de la fiebre tifoidea ha de recordar el médico, dejando aparte lo referente a hipótesis, teorías, consideraciones, detalles de experimentación, etc. etc.

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