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Studio funzionale della transglutaminasi 3 e di p63 nel differenziamento epidermico mediante l'uso di modelli murini
Get PDFRIASSUNTO BREVE
Il differenziamento epidermico prevede la migrazione dei cheratinociti dallo strato basale dell’epidermide a quelli sopra basali fino al raggiungimento dello strato più superficiale dove formano l’involucro corneo. Durante questo processo i futuri corneociti subiscono una serie di cambiamenti nella loro struttura e tali modifiche sono mediate dagli enzimi transglutaminasi. Le transglutaminasi (TGasi) rappresentano una famiglia di enzimi funzionalmente e strutturalmente correlati che catalizzano la reazione di trasferimento acilico Ca2+-dipendente tra il gruppo γ- carbossiamidico di residui di glutammina e il gruppo amminico di varie ammine primarie. In questo modo le transglutaminasi catalizzano la formazione di legami crociati (cross-linking) fra proteine che compongono l’involucro corneo. Quattro sono le TGasi espresse a livello epidermico (TGasi 1, TGasi 2, TGasi 3 e TGasi 5) e tutte, esclusa la TGasi 1, mostrano anche un’attività GTPasica. Nello strato basale dell’epidermide svolge invece una funzione molto importante il gene p63. Quest’ultimo è un membro della famiglia di p53, la cui trascrizione da due differenti promotori origina due diverse proteine:TAp63 che contiene un dominio di transattivazione all’N-terminale e ∆Np63che non lo possiede. Tale dominio è importante per l’attivazione dei geni bersaglio che presentano un motivo di legame per p63 di conseguenza TAp63 è considerato l’attivatore trascrizionale dei geni indotti durante il differenziamento, mentre l’espressione di ∆Np63 potrebbe essere correlata con il mantenimento dell’attività proliferante delle cellule staminali epidermiche localizzate nella membrana basale. Ognuna di queste due isoforme va inoltre incontro a splicing alternativo al C-terminale originando tre differenti varianti di splicing:α, β, γ. Topi p63-/- presentano severe anomalie nella pelle e negli arti.
Lo scopo di questo progetto di ricerca è sia quello di fornire nuove conoscenze sul ruolo svolto dalla TGasi 3 durante il differenziamento epidermico che quello di individuare nuovi geni la cui attività risulta essere regolata da p63. Per quanto riguarda la TGasi 3 abbiamo deciso di realizzare topi knock-out condizionali attraverso l’utilizzo della ricombinasi Cre e dei siti loxP. Tale sistema consente infatti l’eliminazione tessuto-specifica dell’espressione del gene bersaglio. Durante gli esperimenti preliminari per la creazione di tali topi abbiamo inoltre scoperto due nuove varianti di splicing per il gene TGM3. La prima, denominata ∆6∆7, isolata nel topo manca della cisteina catalitica e sia l’attività di cross-linking che quella GTPasica risultano essere compromesse. La variante ∆9∆10, isolata nell’uomo, pur conservando intatta la triade catalitica presenta una bassa attività transamidasica, mentre l’attività GTPasica risulta essere persa. Nella seconda parte di questo lavoro di tesi abbiamo invece identificato un nuovo gene bersaglio denominato Scotin, la cui espressione risulta essere regolata positivamente in vitro dall’isoforma TA, ma non da ∆N. L’espressione di Scotin è localizzata nel reticolo endoplasmatico e nell’involucro nucleare. Questo gene è inoltre in grado di indurre apoptosi attraverso l’induzione di stress del reticolo endoplasmatico. Esperimenti condotti su colture epiteliali primarie (murine ed umane) hanno evidenziato come l’espressione di Scotin durante il differenziamento sia accompagnata, come atteso, dall’incremento dell’isoforma TA e dalla dimuzione di ∆N. In conclusione i nostri dati hanno portato all’isolamento di Scotin come nuovo gene bersaglio indotto da p63 durante il differenziamento epidermico. Tale meccanismo attiva la risposta allo “stress” da parte del reticolo endoplasmatico,di cui Scotin è un induttore, correlando così per la prima volta queste due differenti vie.SHORT ABSTRACT
Epidermal differentiation begins with the migration of keratinocytes from the basal layer and ends with the formation of the cornified layer. Cell proliferation, differentiation and death occur sequentially and each process is characterized by the expression of specific proteins. Transglutaminases (TGases) play an important role during the differentiation program. These enzymes catalyse post-translational modifications of proteins by the formation of isopeptide bonds (a cross-linking reaction). The cross-linked products, often of high molecular mass, are highly resistant to mechanical challenge and proteolytic degradation. In mammals, nine distinct TGase isoenzymes have been identified at the genomic level. Four TGases are expressed in the epidermis (TGase 1, TGase 2, TGase 3 and TGase 5) and the last three TGases show also a GTPase activity. TGM1-/- knock-out mice show severe epidermal abnormalities and they dye soon after birth, while TGM2 -/- knock-out mice dont’show any epidermal abnormality. During the complex mechanism that leads to epidermis formation an important role is played by the action of p63. This transcription factor, belonging to the p53 gene family, is transcribed from two different promoters, generating two classes of proteins: one containing an N-terminal transactivation domain (TA) and another that lacks this domain (∆N). The first isoform is considered the transcriptional inducer whereas ∆N could exert a role of dominant negative. In addition alternative splicing at the 3’ end of both transcripts (TA and ∆N) generates three different C-terminal splicing variants: α, β and γ. Mice p63-/- show severe limb, cranofacial and skin defects and die soon after birth.
The aim of this work is to acquire new knowledges in the complex mechanism that leads to epidermal differentiation thanks to the investigation of TGase 3 and p63 function during the skin process formation. Regarding TGase 3 we plan to create conditional knock-out mice for this ezyme using the Cre/loxP system. During our work we isolated also two novel splicing variants for the TGM3 gene. The first one, isolated in mouse, lacks exons six and seven (∆6∆7), while the second one, isolated in NHEK cells, lacks of exons nine and ten (∆9∆10). The experiment demonstrated that the ∆9∆10 is at least 4 times less active than the wild type one, while ∆6∆7 is inactive because it lacks of the catalytic cysteine Regarding to the GTPase, it is lost in both splicing variants because several residues, that are important for the creation of the GTP pocket, are missed. In the second part of this work, we isolated a new p63-target gene: Scotin. This gene was previously isolated as a p53-inducible proapoptotic gene and the protein is located in the endoplasmic reticulum and in the nuclear membrane. Scotin expression is induced in response to endoplasmic reticulum (ER) stress in a p53 dependent or independent manner and this event lead to apoptosis. RT-qPCR showed that Scotin transcript is positively regulated by TAp63α in vitro, while ∆N isoform inhibits its expression. Western blot experiments, performed on differentiating mouse and human primary keratinocytes, revealed Scotin upregulation following the differentiation program. Immunofluorescence performed on mouse new born skin sections demontrated that Scotin expression is located in the suprabasal layer of epidermis where TAp63, but not ∆Np63 is expressed. In conclusion our data lead to isolation of a new p63 target gene induced during the epithelial differentiation program, a complex process that also involves endoplasmic reticulum stress induction
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
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
Author Under Sail The Imagination of Jack London, 1893-1902
No full textIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Intro -- Title Page -- Copyright Page -- Dedication -- Contents -- Acknowledgments -- Introduction -- 1. Spirit Truth -- 2. From Absorption to Theatricality and Back Again -- 3. "I Will Build a New Present" -- 4. Sons as Authors -- 5. Fathers as Publishers -- 6. The Daughter as Author -- 7. Lovers as Authors -- 8. At Sea with the Family -- 9. Yellow News, Yellow Stories -- 10. The Return Home -- Notes -- Bibliography -- Index -- About Jay WilliamsIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Description based on publisher supplied metadata and other sources.Electronic reproduction. Ann Arbor, Michigan : ProQuest Ebook Central, YYYY. Available via World Wide Web. Access may be limited to ProQuest Ebook Central affiliated libraries
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