1,720,958 research outputs found
The complexity of insulin-like growth factor 1 (IGF-1): development of innovative bio-molecular methods for detection and quantification of IGF-1 protein variants and study of their biological roles in physio-pathological conditions
Full text linkThe Insulin-like growth factor-1 (IGF-1) is a polypeptide growth factor with essential roles in physio-pathological conditions, including cellular growth and differentiation. Secretion of IGF-1 required proper post-translational modifications of IGF-1 prohormones (proIGF-1s), which include furin cleavage and N-glycosylation. This complex mechanism of IGF-1 production determines the presence of an IGF-1 pool composed of several IGF-1 protein variants: unglycosylated and glycosylated proIGF-1s, mature IGF-1, and E-peptides. Chapter I of the Thesis provides a description of the complex molecular mechanism regulating the production of the proIGF-1Es and the currently available methods for detecting and quantifying these IGF-1 protein variants. Particular attention was paid to the recent evidence showing that proIGF-1s are stable intermediate of IGF-1 processing and have specific biological activities. Subsequently, we describe a system to produce a relatively high amount of the glycosylated proIGF-1Ea (glyc_proIGF-1Ea) based on HEK-293 cells stably expressing the human IGF-1Ea isoform. Recombinant components of the IGF-1 pool, were characterized by High Resolution Mass Spectrometry (HRMS). Moreover, we used HRMS to detect the proGF-1Ea and Ea-peptide in the cell culture supernatants of HEK-293 cells overexpressing the IGF-1Ea isoform. The different strategies adopted to increase the glyc_proIGf-1Ea yield and the protein stability were also described. The synthesis of new protein standards corresponding to different IGF-1 isoforms, combined with the use of HRMS technology, may provide a useful tool to characterize and quantify the different components of the IGF-1 poll in complex biological matrices. The II chapter describes the development of a fluorescent- based Western Blot (WB) for the simultaneous detection of the three different proIGF-1s and E-peptides. Specifically, we described the use of two different primary antibodies, which recognize different epitopes of proIGF-1 sequences, and two secondary antibodies conjugated to different fluorophores to develop a multiplex fluorescent WB able to discriminate between the different IGF-1 protein variants. Our results demonstrate the feasibility of simultaneously detecting different isoforms of the same protein using different fluorescence filter combinations. Furthermore, combining antibodies to two different epitopes of the same target protein increased the specificity and reliability of protein detection. In the III chapter, we analyzed the molecular mechanisms regulating the
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production of proIGF-1Ea and the biological effect of IGF-1 on 3D Breast Cancer (BC) spheroids formation. We describe different cell-culture conditions that favor the MCF-7 and MDA-2B-231 spheroids formation and the effects of IGF-1 on spheroids growth, compactness, viability, and gene expression patterns of metabolic and epithelial to mesenchymal transition (EMT) markers. Our results highlight the value of 3D spheroid models to better understand the role of IGF-1 on multiple aspects of tumor progression, including cancer dormancy and EMT. The IV chapter describes the impact of N-glycosylation inhibition on IGF-1 production and IGF-1 receptor (IGF1R) signalling pathway activation in the context of diseases associated with aberrant N-glycosylation such as Congenital Disorder of Glycosylation (CDG). Using muscle cellular models and mice models we demonstrated that N-glycosylation inhibition reduces myoblast fusion and impairs the early stage of the myogenic program in vitro and decreased myogenic markers in mice muscles. Finally, muscle gly_proIGF-1Ea production and IGF1R signalling pathway activation were markedly inhibited after N-glycosylation inhibition. Our results offer new insights that increase understanding of possible impairments of the myogenic differentiation capacity in the pathological context of disorders of N-glycosylation
The Role of miRNAs in Downregulation of PTEN for Glioblastoma Multiforme
Full text linkIn this paper we show, in silico, the role of miRNAs in the post-transcriptional
regulation of the onco suppressor gene PTEN by means of a (stochastic) competitive
model including interactions with miRNAs and other concurrent genes. The model
also covers protein formation and the mechanism going from the protein back to the
miRNAs. The numerical simulations confirm the biologically observed situation that the
increase of miRNAs concentration within the cell results in the lower expression of PTEN,
thus suggesting severe implications in brain tumorigenesis
Fluorescence-Based Multiplex Western Blot to Simultaneously Detect the Insulin-Like Growth Factor-1 (IGF-1) Isoforms
No full text: Insulin-like growth factor-1 (IGF-1) is critical for tissue growth and development. The IGF-1 gene contains six exons and due to alternative splicing three different isoforms might be produced: the IGF-1Ea, Eb, and Ec prohormones (proIGF-1s). These proIGF-1s share the same IGF-1 mature sequence, which is responsible for the IGF-1 receptor binding but differ in their carboxy-terminal extensions called Ea-, Eb-, and Ec-peptides. Several lines of evidence indicate that E-peptides control the intracellular proIGF-1s localization and maturation. Here, we present a multiplex Western blotting system able to simultaneously discriminate and quantify mature IGF-1, proIGF-1s and E-peptides within the same sample. HEK293 cells were transiently transfected with plasmids containing the IGF-1Ea, IGF-1Eb, or IGF-1Ec isoform or an empty vector. Two different primary antibodies, which recognize the mature sequence or the common region of E-peptides, were used to detect IGF-1 isoforms, which were subsequently distinguished with secondary antibodies conjugated to different fluorophores. Our results demonstrate the feasibility of simultaneously detecting different IGF-1 isoforms using two primary antibodies directed against different epitopes of proIGF-1s, combined with fluorescence-conjugated secondary antibodies. Furthermore, this dual-epitope strategy increases the specificity of protein detection, making it a valuable tool for studying the diverse roles of IGF-1 isoforms in biological processes
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe 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
Full text link“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
Full text linkWe 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
Full text linkWe 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
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