1,720,973 research outputs found
The role of P-bodies in LIN28A mediated mRNA decay.
No full textBrezmembranski celični kondenzati, kot so P-telesca, predstavljajo pomemben mehanizem prostorske organizacije znotraj celice, ki omogoča natančno regulacijo izražanja genov na posttranskripcijski ravni, vendar njihova funkcionalna povezava s ključnimi regulatorji celične usode, kot je LIN28A, ostaja premalo raziskana. V magistrskem delu smo proučevali vlogo citoplazemskih procesitnih telesc pri LIN28A-posredovanem uravnavanju stabilnosti mRNA v mišjih zarodnih matičnih celicah. Ker smo želeli določiti interakcijske partnerje LIN28A, smo razvili in optimizirali protokol za ko-imunoprecipitacijo, ki je omogočil učinkovito izolacijo specifičnih interakcij. Ugotovili smo, da pufer z 0,1–1 % Tween 20 in fiziološko koncentracijo soli omogoča stabilno zajetje nativnih kompleksov z minimalnim nespecifičnim ozadjem, pri čemer ena ura inkubacije zadošča za zadosten izplen. Dodatno smo optimizirali postopno mehko elucijo z natrijevim citratom, ki ohranja proteinske komplekse v nativni obliki in je primerna za nadaljnjo proteomsko analizo. Z vključitvijo benzonaze smo pokazali, da večina interakcij ni RNA-odvisnav nekaterih primerih je razgradnja RNA celo omogočila vezavo dodatnih proteinov, kar nakazuje na možen zaviralni vpliv RNA na dostopnost vezavnih mest. Z uporabo tehnologije CRISPR/Cas9 smo z izbrisom gena DDX6 onemogočili tvorbo P-telesc ter nato uvedli inducibilno izražanje proteina LIN28A-GFP. Kvantitativna imunofluorescenčna analiza je potrdila, da se LIN28A kopiči v P-telescih le, če so ta funkcionalno prisotna, kar kaže, da je za relokalizacijo ob diferenciacijskih signalih nujna njihova struktura. Nazadnje smo z imunodetekcijo pokazali, da so P-telesca potrebna za stabilizacijo SOX2, kar neposredno potrjuje, da P-telesca niso le mesta razgradnje RNA, temveč prostorsko organizirana regulatorna središča, nujna za zaščito in stabilizacijo mRNA s pomočjo LIN28A. Rezultati naše raziskave prvič eksperimentalno potrjujejo neposredno vlogo P-telesc pri stabilizaciji specifičnih molekul RNA prek delovanja LIN28A, kar predstavlja pomemben dokaz njihove funkcionalnosti v uravnavanju celične usode. To je ključen prispevek k razumevanju, kako celica prostorsko organizira posttranskripcijsko regulacijo, in hkrati odpira nove možnosti za razvoj terapevtskih pristopov, zlasti na področju regenerativne medicine, diferenciacije in bolezni, povezanih z motnjami v stabilnosti mRNA. Na tej osnovi bodo prihodnje raziskave lahko še natančneje opredelile mehanizme delovanja P-telesc in raziskale njihovo vlogo v različnih fizioloških in patoloških kontekstih.Membraneless cellular condensates, such as P-bodies, represent an important mechanism of spatial organization within the cell, enabling precise regulation of gene expression at the post-transcriptional level. Despite increasing interest in their role, the functional connection between P-bodies and key regulators of cell fate, such as LIN28A, remains insufficiently explored. In this master’s thesis, we investigated the role of cytoplasmic membraneless condensates – P-bodies – in LIN28A-mediated regulation of mRNA stability in mouse embryonic stem cells. To identify interaction partners of LIN28A, we developed and optimized a co-immunoprecipitation protocol that enabled efficient isolation of specific interactions. We found that a buffer containing 0.1–1% Tween 20 and physiological salt concentration allows stable capture of native complexes with minimal nonspecific background, with one hour of incubation being sufficient for adequate yield. Furthermore, we optimized a stepwise gentle elution using sodium citrate, which preserves protein complexes in their native form and is suitable for downstream proteomic analysis. By including benzonase, we demonstrated that most interactions are not RNA-dependentin some cases, RNA degradation even enabled the binding of additional proteins, suggesting a potential inhibitory effect of RNA on the accessibility of binding sites. Using CRISPR/Cas9 technology, we deleted the DDX6 gene to prevent the formation of P-bodies and introduced inducible expression of LIN28A-GFP. Quantitative immunofluorescence analysis confirmed that LIN28A accumulates in P-bodies only when they are functionally present, indicating that their structure is essential for relocalization upon differentiation signals. Finally, through immunodetection, we showed that P-bodies are required for the stabilization of SOX2, directly confirming that P-bodies are not merely sites of RNA degradation, but spatially organized regulatory hubs necessary for the protection and stabilization of mRNA through LIN28A. Our results provide the first experimental confirmation of a direct role for P-bodies in the stabilization of specific RNA molecules via LIN28A, representing a significant demonstration of their functionality in regulating cell fate. Our findings advance the understanding of spatial post-transcriptional regulation and open avenues for therapeutic development in regeneration, differentiation, and mRNA stability–related diseases, while future studies may further clarify P-body functions in diverse biological contexts
Systemic studies of RNA binding proteins in stem cell differentiation and pluripotency
Get PDFWhat mechanisms govern and maintain cell states during the process of differentiation is a pivotal question in science. What factors govern the commitment of developmental progenitors from pluripotent stem cells is a representative example of this question. Studies of transcriptional, signaling and chromatin regulation have been highly instrumental for elucidating mechanisms pluripotency maintenance. Nevertheless, current knowledge falls short in explaining the exit from pluripotency and its coupling to lineage commitment. It is unclear how pluripotency and differentiation become stabilized in a mutually exclusive manner.
Here, I deepen our knowledge concerning post-transcriptional mechanisms in pluripotency-differentiation transition. For this purpose I first characterize by quantitative mass spectrometry the changes that occur in the mRNA bound proteome (RBPome) and identify extensive dynamic rearrangements of the RBPome during early embryonic development, from naive to primed stem cell state and to purified primitive streak progenitors (Chapter I). In parallel I identified developmental post-transcriptional processing landscape and show that the dynamic mRNA binding of the RNA-binding protein TDP-43 is critical in pluripotent stem cells (PSCs) for the choice between self-renewal and differentiation/ pluripotency breakdown (Chapter II). In detail, I discovered that TDP-43 directly regulates an evolutionary conserved switch in alternative polyadenylation (APA) of hundreds of transcripts during early differentiation of mouse and human PSCs. Functional analysis revealed that TDP-43 integrates into pluripotency circuitry by repressing the production of lengthened transcripts of the pluripotency factor SOX2, which is targeted for degradation by miR-21. Furthermore, in pluripotent stem cells TDP-43 also promotes self-renewal by repressing the formation of paraspeckles, membraneless nuclear compartments found only in differentiated cells, by enhancing production of short isoform of the lncRNA NEAT1. Conversely, reduction of TDP-43 during differentiation triggers a short-to-long isoform switch of NEAT1, which polymerizes paraspeckles that in turn recruit TDP-43 and relocalise it away from its other RNA targets. Consistent with this cross-regulation, TDP-43 inhibits differentiation and improves somatic cell reprogramming, while paraspeckles promote early differentiation.
These findings reveal how the exit of pluripotency is regulated by a complex posttranscriptional network, which is functionally independent from lineage choices. Apart from its role in the exit of pluripotency, this cross-regulation between paraspeckles and TDP-43 has implications in cancer and neurodegeneration
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
- …
