47,073 research outputs found

    Relacionando a autofagia e o metabolismo nas células dendríticas plasmacitóides

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    Autophagy is a cellular housekeeping mechanism that degrades long-lived redundant and malfunctioning cellular components. It has a vital role in the immune system, not only for being directly involved in the elimination of pathogens, but also through the functional regulation of immune cells, including dendritic cells (DCs). DCs are phagocytic, antigen presenting cells, which connect the innate response with adaptive immunity, by priming naïve T cells and directing the ensuing polarization. Plasmacytoid DCs (pDCs) are a subpopulation of DC specialized in the rapid production of type I interferon, making them particularly relevant in antiviral responses. Their activation and functional response appear to be strongly dependent on the autophagic flux, which led us to investigate if cellular metabolism could play a role in the regulation of pDC autophagy and, thus, in their function. To that end, a pDC cell line (CAL-1) was treated with three different autophagy inhibitors: Spautin-1, VPS34-IN1 and Bafilomycin A1, followed by Western blot analysis of LC3 lipidation, to confirm autophagy inhibition, and 1H NMR metabolomics. The three drugs efficiently inhibited autophagy and affected the cellular metabolism in a significant manner. Interestingly, the effects of the different inhibitors on the cells metabolic profile were very different. Bafilomycin A1 had the least impact on the cells, while Spautin-1 and VPS34-IN1 had a stronger influence on metabolism, albeit in different directions. While Spautin-1 stimulated glycolysis, impaired the TCA cycle, decreased the levels of amino acids, and increased the levels of branch-chained ketoacids, suggesting mTORC1 activation, VPS34-IN1 caused opposite variations, which suggested AMPK activation. As for Bafilomycin A1, it appeared to induce early endoplasmic reticulum (ER) stress responses, with a mild increase in amino acids, uridine nucleotides, ATP, and NAD+. Overall, this work demonstrated the metabolic responses of pDC cells to autophagy inhibition to be highly dependent on the specific inhibitor considered, raising new questions about the molecular targets and mechanisms involved. Their improved understanding in the future will be key to advance knowledge on pDC biology and functional behaviour.A autofagia é um mecanismo que ajuda na manutenção celular, decompondo componentes celulares desgastos, redundantes ou defeituosos. No sistema imunitário, autofagia desempenha um papel vital, não só estando diretamente envolvida na eliminação de agentes patogénicos, mas também através da regulação funcional das células imunes, incluindo as células dendríticas (DCs). As DCs são células fagocíticas, apresentadoras de antigénios, e incluem uma subpopulação especializada na produção de interferão do tipo I, as células dendríticas plasmacitóides (pDCs), especialmente relevantes na resposta antiviral. A ativação e resposta funcional das pDCs parece ser fortemente dependente do fluxo autofágico, o que nos levou a investigar o possível papel do metabolismo celular na regulação da autofagia e da função destas células. Para esse fim, uma linha celular de pDCs (CAL-1) foi tratada com diferentes inibidores da autofagia: Spautin-1, VPS34-IN1 ou Bafilomicina A1, seguido da análise da lipidação de LC3 por Western blot, para confirmar a inibição da autofagia e análise metabolómica por RMN-1H. Os três compostos inibiram eficazmente a autofagia e alteraram o metabolismo significativamente. No entanto, os seus efeitos foram bastante diferentes. A Bafilomicina A1 foi a que teve o menor impacto nas células, enquanto a Spautin-1 e o VPS34-IN1 tiveram uma maior influência no metabolismo, embora em direções diferentes. A Spautin-1 estimulou a glicólise, inibiu o ciclo do TCA, diminuiu os níveis de aminoácidos e aumentou os níveis de cetoácidos de cadeia ramificada, sugerindo a ativação de mTORC1, enquanto o VPS34-IN1 produziu variações opostas que sugeriram ativação da AMPK. Quanto à Bafilomicina A1, a assinatura metabólica foi consistente com a indução inicial de stress do retículo endoplasmático, consistindo num aumento moderado de aminoácidos, nucleótidos de uridina, ATP e NAD+. Em suma, este trabalho demonstrou uma forte dependência da resposta metabólica das CAL-1 em relação ao inibidor de autofagia considerado, levantando-se novas hipóteses sobre os alvos e mecanismos moleculares envolvidos, cuja compreensão aprofundada permitirá, futuramente, fazer avançar o conhecimento sobre a biologia e o comportamento funcional das pDCs.Mestrado em Bioquímic

    Role of the OPA3 protein in the pathogenesis of neurodegenerative diseases

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    OPA3 è una proteina codificata dal genoma nucleare che, grazie a una sequenza di targeting mitocondriale, viene indirizzata ai mitocondri dopo la sua sintesi. Le mutazioni nel gene OPA3 sono associate a due patologie neurodegenerative: la Sindrome di Costeff, causata da mutazioni recessive, e una forma di atrofia ottica dominante che si manifesta con cataratta e spesso sordità. L’esatta funzione e regolazione della proteina non sono ancora state completamente chiarite, così come la sua localizzazione nella membrana mitocondriale esterna o interna. Lo scopo di questa tesi era quello di fare luce sulla funzione della proteina OPA3, con particolare interesse alla dinamica mitocondriale e all’autofagia, sulla sua localizzazione subcellulare ed infine di definire il meccanismo patogenetico nelle patologie neurodegenerative causate da mutazioni in questo gene. A questo scopo abbiamo utilizzato sia una linea di neuroblastoma silenziata stabilmente per OPA3 che linee cellulari primarie derivate da pazienti. I risultati del presente studio dimostrano che la riduzione di OPA3, indotta nelle cellule del neuroblastoma e presente nei fibroblasti derivati dai pazienti, produce alterazioni nel network mitocondriale con uno sbilanciamento a favore della fusione. Questo fenomeno è probabilmente dovuto all’aumento della forma long della proteina OPA1 che è stato riscontrato in entrambi i modelli cellulari. Inoltre, seppur con direzione apparentemente opposta, in entrambi i modelli abbiamo osservato un’alterata regolazione dell’autofagia. Infine, abbiamo confermato che OPA3 localizza nella membrana mitocondriale interna ed è esposta per gran parte nella matrice. Inoltre, un segnale della proteina è stato trovato anche nelle mitochondrial associated membranes, suggerendo un possibile ruolo di OPA3 nel trasferimento dei lipidi tra i mitocondri e il reticolo endoplasmatico. Abbiamo rilevato un’interazione della proteina OPA3 con l’acido fosfatidico che non era mai stata evidenziata fino ad oggi. Queste osservazioni sono compatibili con le alterazioni della dinamica mitocondriale e la disregolazione dell’autofagia documentate nei modelli studiati.OPA3 is a protein encoded by the nuclear genome and, after its synthesis, is directed to mitochondria by a mitochondrial targeting sequence. Mutations in the OPA3 gene are responsible for two neurodegenerative diseases: Costeff syndrome, caused by recessive mutations, and a form of dominant optic atrophy with cataracts and hearing loss. To date, the exact function and regulation of the protein have not yet been understood, as well as its localization in the outer or inner mitochondrial membrane. This thesis aimed to shed light on the subcellular localization and function of the OPA3 protein, with particular focus on mitochondrial dynamics and autophagy, and to define the pathogenic mechanism of OPA3 mutations causing neurodegenerative diseases. For this purpose, we used neuroblastoma cell line stably silenced for OPA3 and patient-derived primary cell lines. The results of the present study demonstrate that a reduction of OPA3 content, induced in neuroblastoma cells and naturally occurring in patient-derived fibroblasts, produces alterations in the mitochondrial network with an unbalance toward fusion. This phenomenon is probably due to the increase in the long form of the OPA1 protein in both cell models. Furthermore, we observed an altered regulation of autophagy. Finally, we confirmed that OPA3 localizes in the inner mitochondrial membrane and is largely exposed in the matrix. Furthermore, the presence of OPA3 was also found in mitochondrial associated membranes, suggesting a possible role of the protein in lipid transfer between mitochondria and the endoplasmic reticulum. We detected an interaction of the OPA3 protein with phosphatidic acid that had never been shown until now. These observations are compatible with the alterations in mitochondrial dynamics and autophagic dysregulation documented in the models examined

    The impact of aging in mutant ataxin-2 induced neurodegeneration

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    Com o aumento da esperança de vida, cada vez mais aparecem doenças que previamente se desconheciam. Portanto, estas doenças não têm cura definitiva, existindo apenas terapêutica para alívio dos sintomas. As doenças neurodegenerativas inserem-se neste grupo de doenças sem cura. À medida que a idade aumenta, o cérebro é afetado pela diminuição e desregulação de certos mecanismos intra e intercelulares. É o caso da autofagia, cuja função de degradação e reciclagem de proteínas e organelos diminui durante o desenvolvimento. Nisto resulta, a acumulação de proteínas de conformação anormal e na formação de agregados proteicos que se tornam evidentes nos neurónios. As doenças de poliglutaminas são um grupo de nove doenças neurodegenerativas, que engloba a Ataxia Espinocerebelosa do tipo 2 (SCA2). A SCA2 é provocada pela expansão anormal do trato CAG que codifica para uma expansão anormal de glutaminas na proteína responsável pela patologia, ATAXINA-2. Dois achados comuns das doenças de poliglutaminas são a presença de agregados neuronais da proteína mutante nas regiões afetadas do cérebro e a desregulação do mecanismos de degradação proteica, como a autofagia. Este projeto teve como objetivo avaliar o efeito da neurodegeneração causada pela ATAXINA-2 mutante nos murganhos jovens e idosos. O aumento da idade contribuiu para um aumento na formação de agregados de ATAXINA-2 mutante e também para a perda neuronal. Os danos nas células provocados pela proteína mutada conduzem à ativação da resposta inflamatória através do recrutamento de astrócitos e ativação da microglia, que se torna mais acentuado numa idade mais avançada. Além disso, a ATAXINA-2 mutada provoca uma redução da ativação da via apoptótica e da quantidade de histonas e, possivelmente, o recrutamento do PABP para os agregados proteicos. Contudo, não foram observadas alterações nos sistemas autofágicos, mitocondriais, sinápticos e na sensitividade da regulação de nutrientes.With the increase in life expectancy, diseases that were previously unknown have increasingly appear. Therefore, these diseases have no definitive cure, therefore only exists therapy for symptoms relief. Neurodegenerative diseases are part of this group of diseases with no cure. As age increases, the brain is affected by the decrease and dysregulation of certain intra and intercellular mechanisms. This is the case of autophagy; whose function of degradation and recycling proteins and organelles decreases during aging. This results in the accumulation of abnormally shaped proteins and the formation of protein aggregates that become evident in neurons. Polyglutamine diseases are a group of nine neurodegenerative diseases, including type 2 spinocerebellar ataxia (SCA2). SCA2 is caused by the abnormal expansion of the CAG tract that encodes for an abnormal expansion of glutamines in the protein responsible for the pathology, ATAXIN-2. Two common hallmarks of polyglutamine diseases are the presence of neuronal aggregates of the mutant protein in the affected regions of the brain and the dysregulation of protein degradation mechanisms, such as autophagy. This project aimed to evaluate the effect of neurodegeneration caused by the mutant ATAXIN-2 on young and old mice. The increase in age contributed to an increase of the formation of mutant ATAXIN-2 aggregates and also to neuronal loss. Cell damage caused by the mutated protein leads to the activation of the inflammatory response through the recruitment of astrocytes and activation of the microglia, which becomes more pronounced at an older age. In addition, the mutated ATAXIN-2 causes a reduction in the activation of the apoptotic pathway and the number of histones and possibly the recruitment of PABP to protein aggregates. However, no changes were observed in the autophagic, mitochondrial, synaptic systems and in the sensitivity of nutrient regulation

    Fine mapping and characterization of genes involved in maize nitrogen utilization efficiency

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    Maize is one of the most productive crop species on earth. A core component of this productivity comes from maize’s responsiveness to nitrogen fertilizers. Despite their importance in crop production, these fertilizers can place an economic burden on growers and can also have negative environmental and health impacts when applied in excess. In order to mitigate the negative effects of nitrogen fertilizers while also developing maize lines productive enough to feed a growing global population, improvements in nitrogen utilization efficiency (NUtE) is an important component of current and future crop research. Importantly, this research must be conducted in conditions that closely mimic commercial-scale production to ensure that the findings are applicable to modern agricultural settings. A key resource that has facilitated understanding of the genetic components associated with nitrogen utilization efficiency has been the Intermated B73 X Mo17 Recombinant Inbred Lines (IBMRIL) X Illinois High Protein (IHP) mapping population. Previous QTL mapping experiments using this population have identified 9 robust genomic regions associated with NUtE and associated traits. In order to identify candidate genes in additional QTL from the IBMRIL X IHP mapping study, several genetic techniques were utilized. Fine mapping was used to reduce the size of a NUtE QTL on chromosome 1 from 15 Mb to 2.5 Mb. This QTL’s effect on NUtE was replicated using an IBMNIL population containing introgressions of the chromosome 1 QTL genomic region of interest. This region contained 22 annotated genes which included two members of the NRT1.1 nitrate transporter gene family (NRT1.1B and NRT1.1C). The NRT1.1 gene family contains four members: NRT1.1A, NRT1.1B, NRT1.1C and NRT1.1D, of which NRT1.1A and NRT1.1B seen to function as the primary functional homologues based on cross species homology and gene expression data. Gene orthologs of NRT1.1A and NRT1.1B have been shown to play critical roles in nitrogen use traits in rice. A publicly available transposon mediated mutant of NRT1.1A was obtained in order to study its effect on NUtE traits in the field. Consistent reductions in stover nitrogen content and biomass were observed in the NRT1.1A mutant, especially under low nitrogen conditions. In addition, rooting area and rooting depth were reduced within the mutant which is consistent with this gene’s role in nitrogen uptake from the soil and regulation of root architecture. Another strategy to identify candidate genes within the NUtE QTL regions is to utilize a population genetics approach that looks at differences in allele frequencies of genetic markers between heterotic groups. Genomic regions with highly divergent allele frequencies could potentially indicate genes under selection to perform effectively within hybrid lines and may play an important role in modern germplasm. Using a measure of this heterotic stratification, which we deem Delta P, we identified candidate genes within the chromosome 4 and chromosome 9 QTL that were in close proximity to SNPs with regionally high Delta P measures. The gene on chromosome 4 was the phosphate transporter (Pho1;2a) and a null mutant of this gene was obtained and grown in the field to study how NUtE associated traits were affected. Interestingly, stover biomass and stover nitrogen measures were consistently higher in the Pho1;2a mutant when compared to wildtype which could be due to alterations in the nitrate/phosphate response pathway. The chromosome 9 QTL contained a gene known to negatively regulate autophagy and nitrogen use traits in plants (HVA22). Near isogenic lines (NILs) containing HVA22 showed changes in grain protein measures, which is consistent with previous data in maize. In an attempt to use HVA22 to further alter grain protein, a transgene was designed that overexpressed the B73 HVA22 gene using the Gamma Zein promoter and transformed into H99. Transgenic and non-transgenic lines were compared in the field under low and high nitrogen rates as both inbred and hybrids and a consistent significant increase in grain protein concentration was observed. In an effort to develop a targeted mutagenesis protocol to allow for the characterization of additional genes involved in NUtE, a genome-editing pipeline was designed that was compatible with our biolistic transformation system. The first gene-targeted using this pipeline was the maize NADP-MDH gene involved in the C4 photosynthetic pathway. This gene functions at the intersection of carbon and nitrogen balance within the plant and modification of this gene could help inform future efforts to modify this C X N balance. A single homozygous NADP-MDH edited line was developed but died as a T0 due to photosynthetic deficits which prevented further study. Another genome editing experiment was performed that targeted the NRT1.1A, NRT1.1B and NRT1.1C genes using a csy4 mediated multiplex genome editing vector that previously hadn’t been shown to function in maize. Six independent edited lines were generated that contained an array of sequence mutations. One of these lines contained a homozygous edit of the NRT1.1B gene and another line contained homozygous edits of NRT1.1A, NRT1.1B and NRT1.1C. Both mutant lines were shown to reduce transcript abundance of edited genes under certain conditions. These lines were carried forward for further characterization in both greenhouse and field trials. In order to assess the nitrate uptake capacity of these edited lines, their sensitivity to chlorate, which is a herbicide taken up through nitrate transporters, was studied. Both the single and triple NRT1.1 mutants showed decreased sensitivity to chlorate as would be expected given their decreased capacity to take up this herbicide. Nitrate treatment assays were also performed on seedlings of these mutants which showed step wise reductions in plant height as additional NRT1.1 transporters were mutated. Field studies that grew these mutant lines under low and high nitrogen conditions showed similar trends as the greenhouse experiments in that stepwise reduction in nitrogen content and biomass were seen in mutant stover and grain. However, despite these obvious indications of reduced nitrogen uptake capacity in NRT1.1B, this mutant showed phenotypes suggesting altered nitrogen response such as increased tillering and reductions in grain protein concentration. The described research validates the hypothesis that the genetic components of hybrid maize NUtE can be identified if properly designed experiments are conducted. By utilizing the extensive genetic resources available to maize researchers, as well as adapting biotechnology tools such as genome editing, we can continue to narrow the knowledge gap regarding the molecular underpinnings of this important trait. Unfortunately, many of the lines generated in this study impaired the plant's ability to efficiently utilize nitrogen, but hopefully these discoveries enable genetically informed improvements in this trait in the future.U of I OnlyAuthor requested U of Illinois access only (OA after 2yrs) in Vireo ETD syste

    Phytochemicals targeting lncRNAs: A novel direction for neuroprotection in neurological disorders

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    Neurological disorders with various etiologies impacting the nervous system are prevalent in clinical practice. Long non-coding RNA (lncRNA) molecules are functional RNA molecules exceeding 200 nucleotides in length that do not encode proteins, but participate in essential activities. Research indicates that lncRNAs may contribute to the pathogenesis of neurological disorders, and may be potential targets for their treatment. Phytochemicals in traditional Chinese herbal medicine (CHM) have been found to exert neuroprotective effects by targeting lncRNAs and regulating gene expression and various signaling pathways. We aim to establish the development status and neuroprotective mechanism of phytochemicals that target lncRNAs through a thorough literature review. A total of 369 articles were retrieved through manual and electronic searches of PubMed, Web of Science, Scopus and CNKI databases from inception to September 2022. The search utilized combinations of natural products, lncRNAs, neurological disorders, and neuroprotective effects as keywords. The included studies, a total of 31 preclinical trials, were critically reviewed to present the current situation and the progress in phytochemical-targeted lncRNAs in neuroprotection. Phytochemicals have demonstrated neuroprotective effects in preclinical studies of various neurological disorders by regulating lncRNAs. These disorders include arteriosclerotic ischemia-reperfusion injury, ischemic/hemorrhagic stroke, Alzheimer's disease, Parkinson's disease, glioma, peripheral nerve injury, post-stroke depression, and depression. Several phytochemicals exert neuroprotective roles through mechanisms such as anti-inflammatory, antioxidant, anti-apoptosis, autophagy regulation, and antagonism of Aβ-induced neurotoxicity. Some phytochemicals targeted lncRNAs and served a neuroprotective role by regulating microRNA and mRNA expression. The emergence of lncRNAs as pathological regulators provides a novel direction for the study of phytochemicals in CHM. Elucidating the mechanism of phytochemicals regulating lncRNAs will help to identify new therapeutic targets and promote their application in precision medicine

    Grasping nothing: a study of minimal ontologies and the sense of music

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    If music were to have a proper sense – one in which it is truly given – one might reasonably place this in sound and aurality. I contend, however, that no such sense exists; rather, the sense of music takes place, and it does so with the impossible. To this end, this thesis – which is a work of philosophy and music – advances an ontology of the impossible (i.e., it thinks the being of what, properly speaking, can have no being) and considers its implications for music, articulating how ontological aporias – of the event, of thinking the absolute, and of sovereignty’s dismemberment – imply senses of music that are anterior to sound. John Cage’s Silent Prayer, a nonwork he never composed, compels a rerethinking of silence on the basis of its contradictory status of existence; Florian Hecker et al.’s Speculative Solution offers a basis for thinking absolute music anew to the precise extent that it is a discourse of meaninglessness; and Manfred Werder’s [yearn] pieces exhibit exemplarily that music’s sense depends on the possibility of its counterfeiting. Inso-much as these accounts produce musical senses that take the place of sound, they are also understood to be performances of these pieces. Here, then, thought is music’s organon and its instrument

    Establishment of a 7-gene prognostic signature based on oxidative stress genes for predicting chemotherapy resistance in pancreatic cancer

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    Background: Oxidative stress is involved in regulating various biological processes in human cancers. However, the effect of oxidative stress on pancreatic adenocarcinoma (PAAD) remained unclear.Methods: Pancreatic cancer expression profiles from TCGA were downloaded. Consensus ClusterPlus helped classify molecular subtypes based on PAAD prognosis-associated oxidative stress genes. Limma package filtered differentially expressed genes (DEGs) between subtypes. A multi-gene risk model was developed using Lease absolute shrinkage and selection operator (Lasso)-Cox analysis. A nomogram was built based on risk score and distinct clinical features.Results: Consistent clustering identified 3 stable molecular subtypes (C1, C2, C3) based on oxidative stress-associated genes. Particularly, C3 had the optimal prognosis with the greatest mutation frequency, activate cell cycle pathway in an immunosuppressed status. Lasso and univariate cox regression analysis selected 7 oxidative stress phenotype-associated key genes, based on which we constructed a robust prognostic risk model independent of clinicopathological features with stable predictive performance in independent datasets. High-risk group was found to be more sensitive to small molecule chemotherapeutic drugs including Gemcitabine, Cisplatin, Erlotinib and Dasatinib. The 6 of 7 genes expressions were significantly associated with methylation. Survival prediction and prognostic model was further improved through a decision tree model by combining clinicopathological features with RiskScore.Conclusion: The risk model containing seven oxidative stress-related genes may have a greater potential to assist clinical treatment decision-making and prognosis determination

    Pathogenesis and treatment of chronic rhinosinusitis from the perspective of sinonasal epithelial dysfunction

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    BackgroundChronic rhinosinusitis (CRS) is a clinical syndrome primarily characterized by long-term mucosal inflammation of the nasal cavity and sinuses. The pathogenesis of CRS is still unclear due to its high heterogeneity. A number of studies have recently focused on the sinonasal epithelium. Thus, there has been a quantum leap in awareness of the role of the sinonasal epithelium, which is now understood as an active functional organ rather than simply an inert mechanical barrier. Undoubtedly, epithelial dysfunction plays a vital role in the onset and development of CRS.ObjectiveIn this article, we discuss the potential contribution of sinonasal epithelium dysfunction to CRS pathogenesis and explore a few current and developing therapeutic options targeting the sinonasal epithelium.ResultsImpaired mucociliary clearance (MCC) and an abnormal sinonasal epithelial barrier are usually considered to be the main causative factors in CRS. Epithelial-derived bioactive substances, such as cytokines, exosomes, and complements, play a vital role in the regulation of innate and adaptive immunity and contribute to the pathophysiological alterations of CRS. The phenomena of epithelial–mesenchymal transition (EMT), mucosal remodeling, and autophagy observed in CRS offer some novel insights into the pathogenesis of this disease. In addition, existing treatment options targeting disorder of sinonasal epithelium can help to relieve the main symptoms associated with CRS to some extent.ConclusionThe presence of a normal epithelium is fundamental for maintaining homeostasis in the nasal and paranasal sinuses. Here, we describe various aspects of the sinonasal epithelium and highlight the contributions of epithelial dysfunction to CRS pathogenesis. Our review provides sound evidence of the need for in-depth study of the pathophysiological alterations of this disease and for the development of novel epithelium-targeting alternative treatments
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