1,720,976 research outputs found
Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc
No full textHeterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context
Linking pseudouridine synthases to growth, developmentand cell competition
Full text linkEukaryotic pseudouridine synthases direct RNA pseudouridylation and bind H⁄ACA small nucleolar RNA (snoRNAs), which, in turn, may act as precursors of microRNA-like molecules. In humans, loss of pseudouridine
synthase activity causes dyskeratosis congenita (DC), a complex systemic disorder characterized by cancer susceptibility, failures in ribosome biogenesis
and telomere stability, and defects in stem cell formation. Considering the significant interest in deciphering the various molecular consequences of pseudouridine synthase failure, we performed a loss of function analysis of minifly (mfl), the pseudouridine synthase gene of Drosophila, in the wing
disc, an advantageous model system for studies of cell growth and differentiation.
In this organ, depletion of the mfl-encoded pseudouridine synthase causes a severe reduction in size by decreasing both the number and the size of wing cells. Reduction of cell number was mainly attributable to cell death rather than reduced proliferation, establishing that apoptosis plays a
key role in the development of the loss of function mutant phenotype.
Depletion of Mfl also causes a proliferative disadvantage in mosaic tissues that leads to the elimination of mutant cells by cell competition. Intriguingly, mfl silencing also triggered unexpected effects on wing patterning and cell differentiation, including deviations from normal lineage
boundaries, mingling of cells of different compartments, and defects in the formation of the wing margin that closely mimic the phenotype of reduced Notch activity. These results suggest that a component of the pseudouridine
synthase loss of function phenotype is caused by defects in Notch signalling
The coding/non-coding overlapping architecture of the gene encoding the Drosophila pseudouridine synthase.
Full text linkBackground: In eukaryotic cells, each molecule of H/ACA small nucleolar RNA (snoRNA)
assembles with four evolutionarily conserved core proteins to compose a specific
ribonucleoprotein particle. One of the four core components has pseudouridine synthase activity
and catalyzes the conversion of a selected uridine to pseudouridine. Members of the pseudouridine
synthase family are highly conserved. In addition to catalyzing pseudouridylation of target RNAs,
they carry out a variety of essential functions related to ribosome biogenesis and, in mammals, to
telomere maintenance. To investigate further the molecular mechanisms underlying the expression
of pseudouridine synthase genes, we analyzed the transcriptional activity of the Drosophila
member of this family in great detail.
Results: The Drosophila gene for pseudouridine synthase, minifly/Nop60b (mfl), encodes two novel
mRNAs ending at a downstream poly(A) site. One species is characterized only by an extended 3'-
untranslated region (3'UTR), while a minor mRNA encodes a variant protein that represents the
first example of an alternative subform described for any member of the family to date. The rare
spliced variant is detected mainly in females and is predicted to have distinct functional properties.
We also report that a cluster comprising four isoforms of a C/D box snoRNA and two highly
related copies of a small ncRNA gene of unknown function is intron-encoded at the gene-variable
3'UTRs. Because this arrangement, the alternative 3' ends allow mfl not only to produce two
distinct protein subforms, but also to release different ncRNAs. Intriguingly, accumulation of all
these intron-encoded RNAs was found to be sex-biased and quantitatively modulated throughout
development and, within the ovaries, the ncRNAs of unknown function were found not
ubiquitously expressed.
Conclusion: Our results expand the repertoire of coding/non-coding transcripts derived from the
gene encoding Drosophila pseudouridine synthase. This gene exhibits a complex and interlaced
organization, and its genetic information may be expressed as different protein subforms and/or
ncRNAs that may potentially contribute to its biological functions
Problematiche di modellazione strutturale di edifici in muratura esistenti soggetti ad azioni sismiche in relazione all’utilizzo dei software commerciali
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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
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