22,744 research outputs found

    Transcriptome sequencing reveals the effects of circRNA on testicular development and spermatogenesis in Qianbei Ma goats

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    Circular RNAs (circRNAs) play an important role in regulating the mammalian reproductive system, especially testicular development and spermatogenesis. However, their functions in testicular development and spermatogenesis in the Qianbei Ma goat, the Guizhou endemic breed are still unclear. In this study, tissue sectioning and circRNAs transcriptome analysis were conducted to compare the changes of morphology and circular RNAs gene expression profile at four different developmental stages (0Y, 0-month-old; 6Y, 6-month-old; 12Y, 12-month-old; 18Y, 18-month-old). The results showed that the circumferences and area of the seminiferous tubule gradually increased with age, and the lumen of the seminiferous tubule in the testis differentiated significantly. 12,784 circRNAs were detected from testicular tissues at four different developmental stages by RNA sequencing, and 8,140 DEcircRNAs (differentially expressed circRNAs) were found in 0Y vs. 6Y, 6Y vs. 12Y, 12Y vs. 18Y and 0Y vs. 18Y, 0Y vs. 12Y, 6Y vs. 18Y Functional enrichment analysis of the source genes showed that they were mainly enriched in testicular development and spermatogenesis. In addition, the miRNAs and mRNAs associated with DECircRNAs in 6 control groups were predicted by bioinformatics, and 81 highly expressed DECircRNAs and their associated miRNAs and mRNAs were selected to construct the ceRNA network. Through functional enrichment analysis of the target genes of circRNAs in the network, some candidate circRNAs related to testicular development and spermatogenesis were obtained. Such as circRNA_07172, circRNA_04859, circRNA_07832, circRNA_00032 and circRNA_07510. These results will help to reveal the mechanism of circRNAs in testicular development and spermatogenesis, and also provide some guidance for goat reproduction

    Pollution-induced community tolerance in freshwater biofilms – from molecular mechanisms to loss of community functions

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    Exposure to herbicides poses a threat to aquatic biofilms by affecting their community structure, physiology and function. These changes render biofilms to become more tolerant, but on the downside community tolerance has ecologic costs. A concept that addresses induced community tolerance to a pollutant (PICT) was introduced by Blanck and Wängberg (1988). The basic principle of the concept is that microbial communities undergo pollution-induced succession when exposed to a pollutant over a long period of time, which changes communities structurally and functionally and enhancing tolerance to the pollutant exposure. However, the mechanisms of tolerance and the ecologic consequences were hardly studied up to date. This thesis addresses the structural and functional changes in biofilm communities and applies modern molecular methods to unravel molecular tolerance mechanisms. Two different freshwater biofilm communities were cultivated for a period of five weeks, with one of the communities being contaminated with 4 μg L-1 diuron. Subsequently, the communities were characterized for structural and functional differences, especially focusing on their crucial role of photosynthesis. The community structure of the autotrophs was assessed using HPLC-based pigment analysis and their functional alterations were investigated using Imaging-PAM fluorometry to study photosynthesis and community oxygen profiling to determine net primary production. Then, the molecular fingerprints of the communities were measured with meta-transcriptomics (RNA-Seq) and GC-based community metabolomics approaches and analyzed with respect to changes in their molecular functions. The communities were acute exposed to diuron for one hour in a dose-response design, to reveal a potential PICT and uncover related adaptation to diuron exposure. The combination of apical and molecular methods in a dose-response design enabled the linkage of functional effects of diuron exposure and underlying molecular mechanisms based on a sensitivity analysis. Chronic exposure to diuron impaired freshwater biofilms in their biomass accrual. The contaminated communities particularly lost autotrophic biomass, reflected by the decrease in specific chlorophyll a content. This loss was associated with a change in the molecular fingerprint of the communities, which substantiates structural and physiological changes. The decline in autotrophic biomass could be due to a primary loss of sensitive autotrophic organisms caused by the selection of better adapted species in the course of chronic exposure. Related to this hypothesis, an increase in diuron tolerance has been detected in the contaminated communities and molecular mechanisms facilitating tolerance have been found. It was shown that genes of the photosystem, reductive-pentose phosphate cycle and arginine metabolism were differentially expressed among the communities and that an increased amount of potential antioxidant degradation products was found in the contaminated communities. This led to the hypothesis that contaminated communities may have adapted to oxidative stress, making them less sensitive to diuron exposure. Moreover, the photosynthetic light harvesting complex was altered and the photoprotective xanthophyll cycle was increased in the contaminated communities. Despite these adaptation strategies, the loss of autotrophic biomass has been shown to impair primary production. This impairment persisted even under repeated short-term exposure, so that the tolerance mechanisms cannot safeguard primary production as a key function in aquatic systems.:1. The effect of chemicals on organisms and their functions .............................. 1 1.1 Welcome to the anthropocene .......................................................................... 1 1.2 From cellular stress responses to ecosystem resilience ................................... 3 1.2.1 The individual pursuit for homeostasis ....................................................... 3 1.2.2 Stability from diversity ................................................................................. 5 1.3 Community ecotoxicology - a step forward in monitoring the effects of chemical pollution? ................................................................................................................. 6 1.4 Functional ecotoxicological assessment of microbial communities ................... 9 1.5 Molecular tools – the key to a mechanistic understanding of stressor effects from a functional perspective in microbial communities? ...................................... 12 2. Aims and Hypothesis ......................................................................................... 14 2.1 Research question .......................................................................................... 14 2.2 Hypothesis and outline .................................................................................... 15 2.3 Experimental approach & concept .................................................................. 16 2.3.1 Aquatic freshwater biofilms as model community ..................................... 16 2.3.2 Diuron as model herbicide ........................................................................ 17 2.3.3 Experimental design ................................................................................. 18 3. Structural and physiological changes in microbial communities after chronic exposure - PICT and altered functional capacity ................................................. 21 3.1 Introduction ..................................................................................................... 21 3.2 Methods .......................................................................................................... 23 3.2.1 Biofilm cultivation ...................................................................................... 23 3.2.2 Dry weight and autotrophic index ............................................................. 23 3.2.4 Pigment analysis of periphyton ................................................................. 23 3.2.4.1 In-vivo pigment analysis for community characterization ....................... 24 3.2.4.2 In-vivo pigment analysis based on Imaging-PAM fluorometry ............... 24 3.2.4.3 In-vivo pigment fluorescence for tolerance detection ............................. 26 3.2.4.4 Ex-vivo pigment analysis by high-pressure liquid-chromatography ....... 27 3.2.5 Community oxygen metabolism measurements ....................................... 28 3.3 Results and discussion ................................................................................... 29 3.3.1 Comparison of the structural community parameters ............................... 29 3.3.2 Photosynthetic activity and primary production of the communities after selection phase ................................................................................................. 33 3.3.3 Acquisition of photosynthetic tolerance .................................................... 34 3.3.4 Primary production at exposure conditions ............................................... 36 3.3.5 Tolerance detection in primary production ................................................ 37 3.4 Summary and Conclusion ........................................................................... 40 4. Community gene expression analysis by meta-transcriptomics ................... 41 4.1 Introduction to meta-transcriptomics ............................................................... 41 4.2. Methods ......................................................................................................... 43 4.2.1 Sampling and RNA extraction................................................................... 43 4.2.2 RNA sequencing analysis ......................................................................... 44 4.2.3 Data assembly and processing................................................................. 45 4.2.4 Prioritization of contigs and annotation ..................................................... 47 4.2.5 Sensitivity analysis of biological processes .............................................. 48 4.3 Results and discussion ................................................................................... 48 4.3.1 Characterization of the meta-transcriptomic fingerprints .......................... 49 4.3.2 Insights into community stress response mechanisms using trend analysis (DRomic’s) ......................................................................................................... 51 4.3.3 Response pattern in the isoform PS genes .............................................. 63 4.5 Summary and conclusion ................................................................................ 65 5. Community metabolome analysis ..................................................................... 66 5.1 Introduction to community metabolomics ........................................................ 66 5.2 Methods .......................................................................................................... 68 5.2.1 Sampling, metabolite extraction and derivatisation................................... 68 5.2.2 GC-TOF-MS analysis ............................................................................... 69 5.2.3 Data processing and statistical analysis ................................................... 69 5.3 Results and discussion ................................................................................... 70 5.3.1 Characterization of the metabolic fingerprints .......................................... 70 5.3.2 Difference in the metabolic fingerprints .................................................... 71 5.3.3 Differential metabolic responses of the communities to short-term exposure of diuron ............................................................................................................ 73 5.4 Summary and conclusion ................................................................................ 78 6. Synthesis ............................................................................................................. 79 6.1 Approaches and challenges for linking molecular data to functional measurements ...................................................................................................... 79 6.2 Methods .......................................................................................................... 83 6.2.1 Summary on the data ............................................................................... 83 6.2.2 Aggregation of molecular data to index values (TELI and MELI) .............. 83 6.2.3 Functional annotation of contigs and metabolites using KEGG ................ 83 6.3 Results and discussion ................................................................................... 85 6.3.1 Results of aggregation techniques ........................................................... 85 6.3.2 Sensitivity analysis of the different molecular approaches and endpoints 86 6.3.3 Mechanistic view of the molecular stress responses based on KEGG functions ............................................................................................................ 89 6.4 Consolidation of the results – holistic interpretation and discussion ............... 93 6.4.1 Adaptation to chronic diuron exposure - from molecular changes to community effects.............................................................................................. 93 6.4.2 Assessment of the ecological costs of Pollution-induced community tolerance based on primary production ............................................................. 94 6.5 Outlook ............................................................................................................ 9

    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

    Sanfilippo syndrome: molecular basis, disease models and therapeutic approaches

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    Sanfilippo syndrome or mucopolysaccharidosis III is a lysosomal storage disorder caused by mutations in genes responsible for the degradation of heparan sulfate, a glycosaminoglycan located in the extracellular membrane. Undegraded heparan sulfate molecules accumulate within lysosomes leading to cellular dysfunction and pathology in several organs, with severe central nervous system degeneration as the main phenotypical feature. The exact molecular and cellular mechanisms by which impaired degradation and storage lead to cellular dysfunction and neuronal degeneration are still not fully understood. Here, we compile the knowledge on this issue and review all available animal and cellular models that can be used to contribute to increase our understanding of Sanfilippo syndrome disease mechanisms. Moreover, we provide an update in advances regarding the different and most successful therapeutic approaches that are currently under study to treat Sanfilippo syndrome patients and discuss the potential of new tools such as induced pluripotent stem cells to be used for disease modeling and therapy development

    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

    Estudo de alterações genéticas no NAPRT e NAMPT em cancro

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    NAD+ is both a co-enzyme for oxidation-reduction reactions and a substrate for NAD+ -consuming enzymes and therefore, it is critical for many cellular processes. In mammalian cells, intracellular NAD+ can be synthesized through either de novo synthesis, from tryptophan, or via salvage pathways, from other precursors, such as NAM and NA which are converted by NAMPT and NAPRT, respectively. Changes in the NAD+ content in cells and tissues are linked with a wide variety of diseases, including cancer. A large proportion of cancer cases is still diagnosed only at an advanced stage and thus, there is a need for new affordable, specific, sensitive and non-invasive biomarkers. Salivary biomarkers can meet these criteria and thus, are promising tools in cancer screening, diagnostic and prognostic. The main objective of this work was to study genetic alterations in NAMPT and NAPRT in DNA samples from healthy donors and in samples from cancer patients. For this, DNA and RNA extraction from saliva samples was optimized, as a starting point to study this biofluid as a source for cancer biomarkers. The results from the bioinformatics analysis showed that the frequency of alterations in NAPRT and NAMPT genes is low in the cancer contexts investigated. Nevertheless, it is still necessary to further study the impact that these alterations might have. There is also a great need to investigate and optimize methods for saliva studies, in order to promote it as a liquid biopsy of regular use in clinical settings.O NAD+ funciona como coenzima em reações de oxidação-redução e como um substrato para determinadas enzimas e tem, portanto, um papel crítico em muitos processos celulares. Nas células de mamíferos, o NAD+ intracelular pode ser sintetizado através da síntese de novo, a partir do triptofano, ou através das vias de “reciclagem”, a partir de outros precursores, como NAM e NA, que são convertidos por NAMPT e NAPRT, respetivamente. Alterações no conteúdo de NAD+ em células e tecidos estão relacionadas com uma ampla variedade de doenças, incluindo cancro. Uma grande proporção dos casos de tumores ainda é diagnosticada já num estadio avançado e, por isso, são necessários novos biomarcadores economicamente acessíveis, específicos, sensíveis e não invasivos. Os biomarcadores salivares conseguem cumprir esses critérios e são, assim, moléculas promissoras para o rastreio, diagnóstico e prognóstico de cancro. O principal objetivo deste trabalho foi estudar alterações genéticas no NAMPT e NAPRT em amostras de DNA de dadores saudáveis e em amostras de pacientes com cancro. Para isto, foi otimizada a extração de DNA e RNA a partir de amostras de saliva, como ponto de partida para estudar este biofluido como fonte de biomarcadores de cancro. Os resultados da análise bioinformática mostraram que a frequência de alterações nos genes NAPRT e NAMPT é baixa nos contextos de cancro investigados. Ainda assim, serão necessários mais estudos para analisar o impacto que estas alterações poderão ter. Há também uma grande necessidade de investigar e otimizar métodos para estudos em saliva, a fim de promovê-la como biópsia líquida de uso generalizado em ambiente clínico.Mestrado em Biomedicina Molecula

    New functions of platelet C3G: Involvement in TPO-regulation, ischemia-induced angiogenesis and tumor metastasis

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    [ES] Las GTPasas son proteínas que regulan una gran variedad de procesos celulares, entre los que cabe destacar la proliferación, la diferenciación celular y la apoptosis. Estas proteínas alternan entre dos confirmaciones: una activa o unida a GTP y una inactiva o unida a GDP. El intercambio de GDP por GTP esta catalizado por un grupo de proteínas denominadas GEF (factores intercambiadores de nucleótidos de guanina), mientras que las proteínas GAP (proteínas activadoras de la actividad GTPasa) inhiben a la GTPasa. C3G es un GEF para varias GTPasas de la familia de Ras, principalmente de Rap1, R-Ras y TC21, y para una GTPasa de la familia de Rho, TC10. Mediante el uso de modelos animales que expresan de manera específica en plaquetas y megacariocitos o bien C3G (tgC3G), o bien una forma mutante de C3G (caracterizada por la pérdida del dominio catalítico, tgC3GCat), nuestro grupo ha demostrado la participación de C3G en la diferenciación megacariocítica, así como en la regulación de la función hemostática de las plaquetas. En concreto, las plaquetas tgC3G presentan una mayor activación y agregación plaquetaria, que se correlaciona con tiempos de sangrado significativamente inferiores en los ratones tgC3G, además de un incremento en la formación de trombos en modelos in vivo. La sobreexpresión de C3G plaquetario, también genera una alteración en la secreción de los gránulos-α, caracterizada por la retención del factor de crecimiento del endotelio vascular (VEGF) y de trombospondina- 1 (TSP-1) en el citoplasma de las plaquetas, dando lugar a un secretoma netamente proangiogénico. Como resultado de la mayor capacidad proangiogénica de las plaquetas que sobreexpresan C3G, los ratones tgC3G mostraron un crecimiento tumoral más rápido en dos modelos heterotópicos de implantación tumoral. Además, la proteína C3G plaquetaria promueve la metástasis pulmonar de células de melanoma (B16-F10). Sin embargo, la expresión transgénica de C3G no altera los recuentos plaquetarios en sangre periférica. En esta Tesis, hemos profundizado en el papel de C3G en la megacariopoyesis, en la angiogénesis inducida por isquemia y en la metástasis tumoral. Para ello, hemos desarrollado un modelo animal adicional (C3G-KO), en el cual C3G se encuentra específicamente delecionado en megacariocitos (Mk). Al igual que lo observado en ratones tgC3G, los animales C3G-KO tampoco mostraron diferencias ni en el número de Mk en médula ósea, ni en los recuentos plaquetarios en sangre periférica. Sin embargo, la deleción de C3G resultó en una mayor maduración megacariocítica in vitro cuando las médulas óseas fueron cultivas en medio enriquecido con trombopoyetina (TPO) junto con un cocktail de citocinas, sugiriendo un posible papel de C3G en una megacariopoyesis patológica. En base a esto, hemos analizamos el papel de C3G en dos modelos in vivo de megacariopoyesis patológica: la inyección de TPO y la mielosupresión inducida por 5- Fluoruracilo (5-FU). La inyección intravenosa de TPO estimula la megacariopoyesis, incrementando los niveles plaquetarios; mientras que el 5-FU induce la depleción de la médula ósea alrededor del séptimo día tas la inyección, lo que va seguido de un profundo incremento en el recuento plaquetario, proceso conocido como rebote plaquetario (platelet rebound) tras 10-15 días de tratamiento

    RNA pull-down-confocal nanoscanning (RP-CONA), a novel method for studying RNA/protein interactions in cell extracts that detected potential drugs for Parkinson’s disease targeting RNA/HuR complexes

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    MicroRNAs (miRNAs, miRs) are a class of small non-coding RNAs that regulate gene expression through specific base-pair targeting. The functional mature miRNAs usually undergo a two-step cleavage from primary miRNAs (pri-miRs), then precursor miRNAs (pre-miRs). The biogenesis of miRNAs is tightly controlled by different RNA-binding proteins (RBPs). The dysregulation of miRNAs is closely related to a plethora of diseases. Targeting miRNA biogenesis is becoming a promising therapeutic strategy. HuR and MSI2 are both RBPs. MiR-7 is post-transcriptionally inhibited by the HuR/MSI2 complex, through a direct interaction between HuR and the conserved terminal loop (CTL) of pri-miR-7-1. Small molecules dissociating pri-miR-7/HuR interaction may induce miR-7 production. Importantly, the miR-7 levels are negatively correlated with Parkinson’s disease (PD). PD is a common, incurable neurodegenerative disease causing serious motor deficits. A hallmark of PD is the presence of Lewy bodies in the human brain, which are inclusion bodies mainly composed of an aberrantly aggregated protein named α-synuclein (α-syn). Decreasing α-syn levels or preventing α-syn aggregation are under investigation as PD treatments. Notably, α-syn is negatively regulated by several miRNAs, including miR-7, miR-153, miR-133b and others. One hypothesis is that elevating these miRNA levels can inhibit α-syn expression and ameliorate PD pathologies. In this project, we identified miR-7 as the most effective α-syn inhibitor, among the miRNAs that are downregulated in PD, and with α-syn targeting potentials. We also observed potential post-transcriptional inhibition on miR-153 biogenesis in neuroblastoma, which may help to uncover novel therapeutic targets towards PD. To identify miR-7 inducers that benefit PD treatment by repressing α-syn expression, we developed a novel technique RNA Pull-down Confocal Nanoscaning (RP-CONA) to monitor the binding events between pri-miR-7 and HuR. By attaching FITC-pri-miR-7-1-CTL-biotin to streptavidin-coated agarose beads and incubating them in human cultured cell lysates containing overexpressed mCherry-HuR, the bound RNA and protein can be visualised as quantifiable fluorescent rings in corresponding channels in a confocal high-content image system. A pri-miR-7/HuR inhibitor can decrease the relative mCherry/FITC intensity ratio in RP-CONA. With this technique, we performed several small-scale screenings and identified that a bioflavonoid, quercetin can largely dissociate the pri-miR-7/HuR interaction. Further studies proved that quercetin was an effective miR-7 inducer as well as α-syn inhibitor in HeLa cells. To understand the mechanism of quercetin mediated α-syn inhibition, we tested the effects of quercetin treatment with miR-7-1 and HuR knockout HeLa cells. We found that HuR was essential in this pathway, while miR-7 hardly contributed to the α-syn inhibition. HuR can directly bind an AU-rich element (ARE) at the 3’ untranslated region (3’-UTR) of α-syn mRNA and promote translation. We believe quercetin mainly disrupts the ARE/HuR interaction and disables the HuR-induced α-syn expression. In conclusion, we developed and optimised RP-CONA, an on-bead, lysate-based technique detecting RNA/protein interactions, as well as identifying RNA/protein modulators. With RP-CONA, we found quercetin inducing miR-7 biogenesis, and inhibiting α-syn expression. With these beneficial effects, quercetin has great potential to be applied in the clinic of PD treatment. Finally, RP-CONA can be used in many other RNA/protein interactions studies
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