8,599 research outputs found

    OLIG2 neural progenitor cell development and fate in Down syndrome

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    Down syndrome (DS) is caused by triplication of human chromosome 21 (HSA21) and is the most common genetic form of intellectual disability. It is unknown precisely how triplication of HSA21 results in the intellectual disability, but it is thought that the global transcriptional dysregulation caused by trisomy 21 perturbs multiple aspects of neurodevelopment that cumulatively contribute to its etiology. While the characteristics associated with DS can arise from any of the genes triplicated on HSA21, in this work we focus on oligodendrocyte transcription factor 2 (OLIG2). The progeny of neural progenitor cells (NPCs) expressing OLIG2 are likely to be involved in many of the cellular changes underlying the intellectual disability in DS. To explore the fate of OLIG2+ neural progenitors, we took advantage of two distinct models of DS, the Ts65Dn mouse model and induced pluripotent stem cells (iPSCs) derived from individuals with DS. Our results from these two systems identified multiple perturbations in development in the cellular progeny of OLIG2+ NPCs. In Ts65Dn, we identified alterations in neurons and glia derived from the OLIG2 expressing progenitor domain in the ventral spinal cord. There were significant differences in the number of motor neurons and interneurons present in the trisomic lumbar spinal cord depending on age of the animal pointing both to a neurodevelopment and a neurodegeneration phenotype in the Ts65Dn mice. Of particular note, we identified changes in oligodendrocyte (OL) maturation in the trisomic mice that are dependent on spatial location and developmental origin. In the dorsal corticospinal tract, there were significantly fewer mature OLs in the trisomic mice, and in the lateral funiculus we observed the opposite phenotype with more mature OLs being present in the trisomic animals. We then transitioned our studies into iPSCs where we were able to pattern OLIG2+ NPCs to either a spinal cord-like or a brain-like identity and study the OL lineage that differentiated from each progenitor pool. Similar to the region-specific dysregulation found in the Ts65Dn spinal cord, we identified perturbations in trisomic OLs that were dependent on whether the NPCs had been patterned to a brain-like or spinal cord-like fate. In the spinal cord-like NPCs, there was no difference in the proportion of cells expressing either OLIG2 or NKX2.2, the two transcription factors whose co-expression is essential for OL differentiation. Conversely, in the brain-like NPCs, there was a significant increase in OLIG2+ cells in the trisomic culture and a decrease in NKX2.2 mRNA expression. We identified a sonic hedgehog (SHH) signaling based mechanism underlying these changes in OLIG2 and NKX2.2 expression in the brain-like NPCs and normalized the proportion of trisomic cells expressing the transcription factors to euploid levels by modulating the activity of the SHH pathway. Finally, we continued the differentiation of the brain-like and spinal cord-like NPCs to committed OL precursor cells (OPCs) and allowed them to mature. We identified an increase in OPC production in the spinal cord-like trisomic culture which was not present in the brain-like OPCs. Conversely, we identified a maturation deficit in the brain-like trisomic OLs that was not present in the spinal cord-like OPCs. These results underscore the importance of regional patterning in characterizing changes in cell differentiation and fate in DS. Together, the findings presented in this work contribute to the understanding of the cellular and molecular etiology of the intellectual disability in DS and in particular the contribution of cells differentiated from OLIG2+ progenitors

    A direct-laser-written heart-on-a-chip platform for generation and stimulation of engineered heart tissues

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    In this dissertation, we first develop a versatile microfluidic heart-on-a-chip model to generate 3D-engineered human cardiac microtissues in highly-controlled microenvironments. The platform, which is enabled by direct laser writing (DLW), has tailor-made attachment sites for cardiac microtissues and comes with integrated strain actuators and force sensors. Application of external pressure waves to the platform results in controllable time-dependent forces on the microtissues. Conversely, oscillatory forces generated by the microtissues are transduced into measurable electrical outputs. After characterization of the responsivity of the transducers, we demonstrate the capabilities of this platform by studying the response of cardiac microtissues to prescribed mechanical loading and pacing. Next, we tune the geometry and mechanical properties of the platform to enable parametric studies on engineered heart tissues. We explore two geometries: a rectangular seeding well with two attachment sites, and a stadium-like seeding well with six attachment sites. The attachment sites are placed symmetrically in the longitudinal direction. The former geometry promotes uniaxial contraction of the tissues; the latter additionally induces diagonal fiber alignment. We systematically increase the length for both configurations and observe a positive correlation between fiber alignment at the center of the microtissues and tissue length. However, progressive thinning and “necking” is also observed, leading to the failure of longer tissues over time. We use the DLW technique to improve the platform, softening the mechanical environment and optimizing the attachment sites for generation of stable microtissues at each length and geometry. Furthermore, electrical pacing is incorporated into the platform to evaluate the functional dynamics of stable microtissues over the entire range of physiological heart rates. Here, we typically observe a decrease in active force and contraction duration as a function of frequency. Lastly, we use a more traditional ?TUG platform to demonstrate the effects of subthreshold electrical pacing on the rhythm of the spontaneously contracting cardiac microtissues. Here, we observe periodic M:N patterns, in which there are ? cycles of stimulation for every ? tissue contractions. Using electric field amplitude, pacing frequency, and homeostatic beating frequencies of the tissues, we provide an empirical map for predicting the emergence of these rhythms

    Estudo da remodelagem reversa miocárdica através da análise proteómica do miocárdio e do líquido pericárdico

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    Valve replacement remains as the standard therapeutic option for aortic stenosis patients, aiming at abolishing pressure overload and triggering myocardial reverse remodeling. However, despite the instant hemodynamic benefit, not all patients show complete regression of myocardial hypertrophy, being at higher risk for adverse outcomes, such as heart failure. The current comprehension of the biological mechanisms underlying an incomplete reverse remodeling is far from complete. Furthermore, definitive prognostic tools and ancillary therapies to improve the outcome of the patients undergoing valve replacement are missing. To help abridge these gaps, a combined myocardial (phospho)proteomics and pericardial fluid proteomics approach was followed, taking advantage of human biopsies and pericardial fluid collected during surgery and whose origin anticipated a wealth of molecular information contained therein. From over 1800 and 750 proteins identified, respectively, in the myocardium and in the pericardial fluid of aortic stenosis patients, a total of 90 dysregulated proteins were detected. Gene annotation and pathway enrichment analyses, together with discriminant analysis, are compatible with a scenario of increased pro-hypertrophic gene expression and protein synthesis, defective ubiquitinproteasome system activity, proclivity to cell death (potentially fed by complement activity and other extrinsic factors, such as death receptor activators), acute-phase response, immune system activation and fibrosis. Specific validation of some targets through immunoblot techniques and correlation with clinical data pointed to complement C3 β chain, Muscle Ring Finger protein 1 (MuRF1) and the dual-specificity Tyr-phosphorylation regulated kinase 1A (DYRK1A) as potential markers of an incomplete response. In addition, kinase prediction from phosphoproteome data suggests that the modulation of casein kinase 2, the family of IκB kinases, glycogen synthase kinase 3 and DYRK1A may help improve the outcome of patients undergoing valve replacement. Particularly, functional studies with DYRK1A+/- cardiomyocytes show that this kinase may be an important target to treat cardiac dysfunction, provided that mutant cells presented a different response to stretch and reduced ability to develop force (active tension). This study opens many avenues in post-aortic valve replacement reverse remodeling research. In the future, gain-of-function and/or loss-of-function studies with isolated cardiomyocytes or with animal models of aortic bandingdebanding will help disclose the efficacy of targeting the surrogate therapeutic targets. Besides, clinical studies in larger cohorts will bring definitive proof of complement C3, MuRF1 and DYRK1A prognostic value.A substituição da válvula aórtica continua a ser a opção terapêutica de referência para doentes com estenose aórtica e visa a eliminação da sobrecarga de pressão, desencadeando a remodelagem reversa miocárdica. Contudo, apesar do benefício hemodinâmico imediato, nem todos os pacientes apresentam regressão completa da hipertrofia do miocárdio, ficando com maior risco de eventos adversos, como a insuficiência cardíaca. Atualmente, os mecanismos biológicos subjacentes a uma remodelagem reversa incompleta ainda não são claros. Além disso, não dispomos de ferramentas de prognóstico definitivos nem de terapias auxiliares para melhorar a condição dos pacientes indicados para substituição da válvula. Para ajudar a resolver estas lacunas, uma abordagem combinada de (fosfo)proteómica e proteómica para a caracterização, respetivamente, do miocárdio e do líquido pericárdico foi seguida, tomando partido de biópsias e líquidos pericárdicos recolhidos em ambiente cirúrgico. Das mais de 1800 e 750 proteínas identificadas, respetivamente, no miocárdio e no líquido pericárdico dos pacientes com estenose aórtica, um total de 90 proteínas desreguladas foram detetadas. As análises de anotação de genes, de enriquecimento de vias celulares e discriminativa corroboram um cenário de aumento da expressão de genes pro-hipertróficos e de síntese proteica, um sistema ubiquitina-proteassoma ineficiente, uma tendência para morte celular (potencialmente acelerada pela atividade do complemento e por outros fatores extrínsecos que ativam death receptors), com ativação da resposta de fase aguda e do sistema imune, assim como da fibrose. A validação de alguns alvos específicos através de immunoblot e correlação com dados clínicos apontou para a cadeia β do complemento C3, a Muscle Ring Finger protein 1 (MuRF1) e a dual-specificity Tyr-phosphoylation regulated kinase 1A (DYRK1A) como potenciais marcadores de uma resposta incompleta. Por outro lado, a predição de cinases a partir do fosfoproteoma, sugere que a modulação da caseína cinase 2, a família de cinases do IκB, a glicogénio sintase cinase 3 e da DYRK1A pode ajudar a melhorar a condição dos pacientes indicados para intervenção. Em particular, a avaliação funcional de cardiomiócitos DYRK1A+/- mostraram que esta cinase pode ser um alvo importante para tratar a disfunção cardíaca, uma vez que os miócitos mutantes responderam de forma diferente ao estiramento e mostraram uma menor capacidade para desenvolver força (tensão ativa). Este estudo levanta várias hipóteses na investigação da remodelagem reversa. No futuro, estudos de ganho e/ou perda de função realizados em cardiomiócitos isolados ou em modelos animais de banding-debanding da aorta ajudarão a testar a eficácia de modular os potenciais alvos terapêuticos encontrados. Além disso, estudos clínicos em coortes de maior dimensão trarão conclusões definitivas quanto ao valor de prognóstico do complemento C3, MuRF1 e DYRK1A.Programa Doutoral em Biomedicin

    Targeting Fusion Proteins of HIV-1 and SARS-CoV-2

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    Viruses are disease-causing pathogenic agents that require host cells to replicate. Fusion of host and viral membranes is critical for the lifecycle of enveloped viruses. Studying viral fusion proteins can allow us to better understand how they shape immune responses and inform the design of therapeutics such as drugs, monoclonal antibodies, and vaccines. This thesis discusses two approaches to targeting two fusion proteins: Env from HIV-1 and S from SARS-CoV-2. The first chapter of this thesis is an introduction to viruses with a specific focus on HIV-1 CD4 mimetic drugs and antibodies against SARS-CoV-2. It discusses the architecture of these viruses and fusion proteins and how small molecules, peptides, and antibodies can target these proteins successfully to treat and prevent disease. In addition, a brief overview is included of the techniques involved in structural biology and how it has informed the study of viruses. For the interested reader, chapter 2 contains a review article that serves as a more in-depth introduction for both viruses as well as how the use of structural biology has informed the study of viral surface proteins and neutralizing antibody responses to them. The subsequent chapters provide a body of work divided into two parts. The first part in chapter 3 involves a study on conformational changes induced in the HIV-1 Env protein by CD4-mimemtic drugs using single particle cryo-EM. The second part encompassing chapters 4 and 5 includes two studies on antibodies isolated from convalescent COVID-19 donors. The former involves classification of antibody responses to the SARS-CoV-2 S receptor-binding domain (RBD). The latter discusses an anti-RBD antibody class that binds to a conserved epitope on the RBD and shows cross-binding and cross-neutralization to other coronaviruses in the sarbecovirus subgenus.</p

    Towards A Graphene Chip System For Blood Clotting Disease Diagnostics

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    Point of care diagnostics (POCD) allows the rapid, accurate measurement of analytes near to a patient. This enables faster clinical decision making and can lead to earlier diagnosis and better patient monitoring and treatment. However, despite many prospective POCD devices being developed for a wide range of diseases this promised technology is yet to be translated to a clinical setting due to the lack of a cost-effective biosensing platform.This thesis focuses on the development of a highly sensitive, low cost and scalable biosensor platform that combines graphene with semiconductor fabrication tech-niques to create graphene field-effect transistors biosensor. The key challenges of designing and fabricating a graphene-based biosensor are addressed. This work fo-cuses on a specific platform for blood clotting disease diagnostics, but the platform has the capability of being applied to any disease with a detectable biomarker.Multiple sensor designs were tested during this work that maximised sensor ef-ficiency and costs for different applications. The multiplex design enabled different graphene channels on the same chip to be functionalised with unique chemistry. The Inverted MOSFET design was created, which allows for back gated measurements to be performed whilst keeping the graphene channel open for functionalisation. The Shared Source and Matrix design maximises the total number of sensing channels per chip, resulting in the most cost-effective fabrication approach for a graphene-based sensor (decreasing cost per channel from £9.72 to £4.11).The challenge of integrating graphene into a semiconductor fabrication process is also addressed through the development of a novel vacuum transfer method-ology that allows photoresist free transfer. The two main fabrication processes; graphene supplied on the wafer “Pre-Transfer” and graphene transferred after met-allisation “Post-Transfer” were compared in terms of graphene channel resistance and graphene end quality (defect density and photoresist). The Post-Transfer pro-cess higher quality (less damage, residue and doping, confirmed by Raman spec-troscopy).Following sensor fabrication, the next stages of creating a sensor platform involve the passivation and packaging of the sensor chip. Different approaches using dielec-tric deposition approaches are compared for passivation. Molecular Vapour Deposi-tion (MVD) deposited Al2O3 was shown to produce graphene channels with lower damage than unprocessed graphene, and also improves graphene doping bringing the Dirac point of the graphene close to 0 V. The packaging integration of microfluidics is investigated comparing traditional soft lithography approaches and the new 3D printed microfluidic approach. Specific microfluidic packaging for blood separation towards a blood sampling point of care sensor is examined to identify the laminar approach for lower blood cell count, as a method of pre-processing the blood sample before sensing.To test the sensitivity of the Post-Transfer MVD passivated graphene sensor de-veloped in this work, real-time IV measurements were performed to identify throm-bin protein binding in real-time on the graphene surface. The sensor was function-alised using a thrombin specific aptamer solution and real-time IV measurements were performed on the functionalised graphene sensor with a range of biologically relevant protein concentrations. The resulting sensitivity of the graphene sensor was in the 1-100 pg/ml concentration range, producing a resistance change of 0.2% per pg/ml. Specificity was confirmed using a non-thrombin specific aptamer as the neg-ative control. These results indicate that the graphene sensor platform developed in this thesis has the potential as a highly sensitive POCD. The processes developed here can be used to develop graphene sensors for multiple biomarkers in the future

    Development of in-vitro in-silico technologies for modelling and analysis of haematological malignancies

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    Worldwide, haematological malignancies are responsible for roughly 6% of all the cancer-related deaths. Leukaemias are one of the most severe types of cancer, as only about 40% of the patients have an overall survival of 10 years or more. Myelodysplastic Syndrome (MDS), a pre-leukaemic condition, is a blood disorder characterized by the presence of dysplastic, irregular, immature cells, or blasts, in the peripheral blood (PB) and in the bone marrow (BM), as well as multi-lineage cytopenias. We have created a detailed, lineage-specific, high-fidelity in-silico erythroid model that incorporates known biological stimuli (cytokines and hormones) and a competing diseased haematopoietic population, correctly capturing crucial biological checkpoints (EPO-dependent CFU-E differentiation) and replicating the in-vivo erythroid differentiation dynamics. In parallel, we have also proposed a long-term, cytokine-free 3D cell culture system for primary MDS cells, which was firstly optimized using easily-accessible healthy controls. This system enabled long-term (24-day) maintenance in culture with high (>75%) cell viability, promoting spontaneous expansion of erythroid phenotypes (CD71+/CD235a+) without the addition of any exogenous cytokines. Lastly, we have proposed a novel in-vitro in-silico framework using GC-MS metabolomics for the metabolic profiling of BM and PB plasma, aiming not only to discretize between haematological conditions but also to sub-classify MDS patients, potentially based on candidate biomarkers. Unsupervised multivariate statistical analysis showed clear intra- and inter-disease separation of samples of 5 distinct haematological malignancies, demonstrating the potential of this approach for disease characterization. The work herein presented paves the way for the development of in-vitro in-silico technologies to better, characterize, diagnose, model and target haematological malignancies such as MDS and AML.Open Acces

    Biomarcadores salivares no cancro da cabeça e pescoço

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    Head and neck cancer (HNC) are a group of cancers which occur in the organs from the anatomical area referred. The HNC staging system is generalized for most subtypes of HNC and restricted for the seven classifying stages of this cancer, which affects the diagnosis and, therefore, the treatment. The possible biomarkers identified for HNC are either non-specific or still waiting for clinical validations. Exosomes are nanovesicles which reflect the molecular composition of the cell of origin, including tumour cells, and can be found in biofluids as saliva, a proximal and easily accessible fluid for HNC. As exosome’ purification methods are being developed, the study of the proteome of salivary exosomes of HNC patients is a promising approach for identification of specific biomarkers for HNC and early diagnosis and prognosis of the cancer. In this thesis project, unstimulated saliva was collected from fifteen HNC patients and three healthy subjects and exosomes were extracted. Protein quantification was performed, and the proteins were analysed by LC-MS, after protein separation with SDS-PAGE and in-gel trypsin digestion. The proteomes of salivary exosomes of HNC demonstrated to be involved in a panoply of biological processes mainly focused in exocytosis, inflammatory response and tumour-related processes, which were expected in this study. The overexpression of α-amylase and immunoglobulin A in salivary exosomes of HNC, when compared with the healthy controls, is considered as a potential HNC biomarker. From the several proteins exclusively in either sample 1, 2, 8, 10, 14 and 15, suprabasin, protein S100-A7, S100-P and cathepsin B were already described as HNC possible biomarker in another type of samples. This study demonstrated that the salivary exosomes are capable to reflect the pathophysiological status of HNC. Furthermore, overexpression of α-amylase and immunoglobulin A in salivary exosomes may be a non-specific biomarker of HNC and the previously described HNC biomarkers need to be further studied in order to understand in which conditions they are expressed.O cancro da cabeça e pescoço (CCP) corresponde a um grupo de cancros que ocorrem nos órgãos da área anatómica referida. O sistema de classificação do CCP é generalizado para a maioria dos subtipos de CCP e restrito aos sete estágios de classificação desse cancro, o que afeta o diagnóstico e, portanto, o tratamento. Os possíveis biomarcadores identificados para CCP ou não são específicos, ou ainda aguardam validações clínicas. Os exossomas são nanovesículas que refletem a composição molecular da sua célula de origem, inclusive células tumorais, e podem ser encontrados em bio fluídos como saliva, um fluído proximal e de fácil acesso. À medida que os métodos de purificação dos exossomas estão a ser desenvolvidos, o estudo do proteoma de exossomas salivares de pacientes com CCP surge como uma abordagem promissora para a identificação de biomarcadores específicos para o mesmo, e diagnóstico e prognóstico precoces do cancro. Neste projeto de tese, a saliva não estimulada foi recolhida de quinze pacientes e três sujeitos saudáveis com CCP e os exossomas foram extraídos. A quantificação de proteínas foi realizada e as proteínas foram analisadas por LC-MS, após separação das proteínas com SDS-PAGE e digestão com tripsina em gel. Os proteomas dos exossomas salivares do HNC demonstraram estar envolvidos numa panóplia de processos biológicos direcionados principalmente para exocitose, resposta inflamatória e processos relacionados a tumores, algo esperado neste estudo. A sobre expressão de α-amilase e imunoglobulina A nos exossomas salivares de HNC, quando comparada com os controlos saudáveis, é considerada um potencial biomarcador de CCP. Das várias proteínas exclusivas nas amostras 1, 2, 8, 10, 14 e 15, a suprabasina, a proteína S100-A7, S100-P e a catepsina B já foram descritas como possíveis biomarcadores do HNC noutro tipo de amostras. Assim, este estudo demonstrou que os exossomas salivares são capazes de refletir o estatuto fisiopatológico do HNC e que a sobre expressão de α-amilase e imunoglobulina A em exossomas salivares pode ser um biomarcador não específico de HNC. Relativamente aos restantes biomarcadores de HNC descritos serão necessários mais estudos para entender em que condições são expressos.Mestrado em Bioquímic

    Uso de las histonas circulantes y sus modificaciones post-traduccionales como biomarcadores en sepsis y shock séptico

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    La sepsis es una afección potencialmente mortal causada por una respuesta anormal del huésped a una infección, produciendo respuestas fisiológicas alteradas que dañan los propios tejidos del paciente y pueden provocar disfunción orgánica e incluso la muerte. Asimismo, algunos pacientes sépticos progresan a shock séptico, caracterizado por alteraciones circulatorias, celulares y metabólicas sustanciales que aumentan el riesgo de mortalidad. A pesar de que la sepsis se caracteriza por un mal funcionamiento del sistema inmunológico, lo que a su vez conduce a una respuesta inmune alterada e inmunosupresión, la alta complejidad de la fisiopatología de la sepsis requiere una mayor investigación para comprender las respuestas inmunes que ocurren durante la sepsis. Asimismo, las histonas extracelulares circulantes han ganado relevancia como mediadores citotóxicos en la sepsis, ya que actúan como patrones moleculares asociados a daño, que inducen estrés oxidativo y activan el inflamasoma NLRP3. Estos mecanismos median la activación de la piroptosis, un mecanismo de muerte celular programada que produce inflamación mediante la expresión de IL-18, IL-1β and IL-1α. Sin embargo, a pesar de la evidencia de activación del inflamasoma en las células inmunes durante la sepsis, se desconoce si las histonas extracelulares son capaces de activar los inflamasomas endoteliales y sus consecuencias. En este trabajo destacamos el papel previamente desconocido de las histonas extracelulares, mediando la activación del inflamasoma NLRP3 y la piroptosis en las células endoteliales, contribuyendo a la disfunción endotelial y la desregulación de la respuesta inmune mediada por el endotelio. Asimismo, también demostramos cómo la acetilación de histonas disminuye la activación de la piroptosis. Además, demostramos que la piroptosis se produce en pacientes con shock séptico y los niveles de histonas circulantes se correlacionan con la expresión de citoquinas proinflamatorias y citoquinas piroptóticas, la liberación de factores de adhesión endotelial y la gravedad de la enfermedad. Proponemos la piroptosis mediada por histonas como un nuevo objetivo para desarrollar intervenciones clínicas. De manera similar, hemos analizado las respuestas inmunorelacionadas que ocurren durante las primeras etapas de la sepsis con el objetivo de proporcionar nuevos datos comparando las cantidades de citoquinas, inmunomoduladores y otros mediadores endoteliales en pacientes críticamente enfermos no sépticos, sépticos y de shock séptico. Nuestro enfoque ayudará a caracterizar rápidamente las respuestas inmunes alteradas en pacientes sépticos y de shock séptico ingresados en la Unidad de Cuidados Intensivos. Finalmente analizamos el papel de la metilación del ADN en el control del sistema inmune séptico. Nuestros resultados demostraron el papel central de la metilación del ADN modulando la respuesta molecular en los pacientes de shock séptico y contribuyendo a la inmunosupresión, a través de la alteración de los patrones de metilación de los promotores de IL-10 y TREM-2.Sepsis is a life-threatening condition caused by an abnormal host response to an infection that produce altered physiological responses which damages own tissues of the patient and can result in organ dysfunction and in some cases death. Likewise, a subset of septic patients progresses to septic shock, characterized by substantial circulatory, cellular and metabolic abnormalities, which substantially increase the risk of mortality. Sepsis is characterized by a malfunction of the immune system and it can lead to an altered immune response and immunosuppression. Moreover, the high complexity of the pathophysiology of sepsis requires of further investigation to characterize the immune responses in sepsis and septic shock. Likewise, circulating extracellular histones have gained relevance as cytotoxic mediators in sepsis pathophysiology, since they act as damage-associated molecular patterns, which induce oxidative stress and activate NLRP3 inflammasome. Subsequently, inflammasome mediates pyroptosis activation, a programmed cell death mechanism that produces inflammation through the release of IL-18, IL-1β and IL-1α. However, despite inflammasome activation may occur in immune cells during sepsis, it is unknown if this process also takes place in endothelial cells and particularly whether extracellular histones are capable of activating endothelial inflammasomes and their consequences. In this work we highlight a previously unknown role for extracellular histones, that mediates the activation of NLRP3 inflammasome and pyroptosis in endothelial cells by contributing to endothelial dysfunction and the dysregulation of the immune response mediated by endothelium. Likewise, we demonstrated how histone acetylation decreases pyroptosis activation. Furthermore, we show how pyroptosis occurs in septic shock patients and how circulating histone levels correlate with the expression of pro-inflammatory and pyroptotic cytokines, the release of endothelial adhesion factors and septic shock severity. We propose histone-mediated pyroptosis as a new target to develop clinical interventions. Similarly, we have analyzed the immune-related responses occurring during the early stages of sepsis with the aim of providing new data by comparing the amounts of cytokines, immune modulators and other endothelial mediators in critically-ill non-septic patients, septic and septic shock patients. Our approach will help to rapidly characterize the altered immune responses in septic and septic shock patients admitted in the Intensive Care Unit. Finally, we also analyzed the role of DNA methylation in the control of septic immune system. Our results demonstrated the central role of DNA methylation modulating the molecular response in septic shock patients and contributing to immunosuppression, through the alteration of DNA methylation patterns of IL-10 and TREM2 promoters

    A novel role for PRC2 activity in light signalling in Arabidopsis thaliana

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    Light is the major regulator of plant development, controlling a number of key stages throughout the plant life cycle. One of these is the transition from skotomorphogenesis to photomorphogenesis in seedlings upon exposure to light. Perception of light via a suite of photoreceptors initiates a massive reorganisation at the transcriptional, translational and post-translational levels across the seedling. In recent years, increasing evidence has also been presented showing epigenetic changes also play a crucial role in this process. The work in this thesis seeks to contribute to this growing field by identifying that the Polycomb Repressive Complex 2 (PRC2) plays a key role in photomorphogenesis via the deposition of H3K27me3, mediated by the histone methyltransferase CURLYLEAF (CLF). Phenotypic and genetic analysis shows that CLF activity is upregulated by blue light in a CRY1-dependent manner, and that loss of CLF leads to elongated hypocotyls – a classic phenotypic trait which has been long used to identify deficiencies in light signalling. RNA-seq and ChIP-qPCR analysis indicates that CLF inhibits hypocotyl elongation via the deposition of the silencing chromatin modification H3K27me3 at expansins and XTH genes. It is also shown that CLF further contributes to the enhancement of auxin signalling in cotyledons via the H3K27me3-mediated silencing of AUX/IAA and GH3 genes. Additional phenotypic analysis indicates that CLF also plays a role in other auxin-regulated processes such as phototropism. Finally, a candidate interaction is identified between CLF and the skotomorphogenesis-promoting transcription factor PIF4, indicating an intriguing possible mechanism for CLF regulation during light responses
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