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Development of viral vector-mediated RNA interference targeting several P2X receptors in the rat central nervous system
Get PDFL’RNA interference (RNAi) e’ un meccanismo di silenziamento genico
sequenza-specifico, conservato durante il processo evolutivo. Il meccanismo di
silenziamento e’ attivato attraverso la degradazione del mRNA complementare o
mediante inibizione del processo trascrittivo o di traduzione. Nelle cellule di
mammifero, l’RNAi viene indotto attraverso l’introduzione o l’espressione
intracellulare di una molecola di RNA a doppio filamento di 20-22 nucleotidi (chiamata
short interfering RNA o siRNA) specifica per l’mRNA bersaglio. Nel passato il
processo di ricombinazione omologa in cellule embrionali era il meccanismo piu’
utilizzato per studiare la funzione di un gene di interesse in mammiferi, mediante
rimozione o sovraespressione del gene stesso. Questo processo risulta pero’ costoso e
non applicabile a molti organismi, inoltre la delezione del gene in esame puo’ portare a
processi di compensazione genica o a fenotipi letali. Attualmente l’RNAi e’
ampiamente impiegato per determinare la funzione di un gene bersaglio sia in sistemi in
vitro che in vivo grazie alla sua elevata versatilita’ ed efficienza ed e’ inoltre utilizzato
nella ricerca farmaceutica per selezionare potenziali bersagli farmacologici.
Questo progetto di ricerca e’ stato sviluppato allo scopo di investigare
l’applicabilita’ del meccanismo di RNA interference per il silenziamento genico nel
sistema nervoso centrale di ratto sia in vitro che in vivo. In particolare, il suo impiego
nel processo di validazione di un gene d’interesse e’ stato analizzato in relazione al
recettore purinergico P2X7, un gene potenzialmente coinvolto in patologie psichiatriche
ed ai due recettori correlati P2X2 e P2X4.
I recettori purinergici P2X2, P2X4 e P2X7 sono membri della famiglia di
recettori-canali cationici attivati dall’ATP, permeabili al sodio, potassio e calcio. Questi
recettori sono espressi in diverse aree cerebrali nei neuroni, dove sono coinvolto nel
rilascio di neurotransmettitori e nell’attivazione di meccanismi multipli di trasduzione
del segnale intracellulare. Il recettore P2X7 risulta inoltre espresso in diverse tipologie
di cellule gliali, in cui e’ coinvolto nella modulazione dei processi neuroinfiammatori e
nel rilascio di diverse citochine. Studi farmacologici e di associazione genetica
suggeriscono un ruolo potenziale del recettore P2X7 nei disordini psichiatrici, inclusi la
depressione unipolare e bipolare. Nel topo l’inibizione dell’mRNA per P2X7, attraverso
RNA antisenso, induce un comportamento di tipo depressivo. Studi di associazione
genetica indicano una correlazione tra specifici polimorfismi nel gene codificante il
recettore P2X7 e i disordine bipolari (Barden et al., 2004 e 2006). I geni per i recettori
P2X2 e P2X4 sono all’interno della stessa regione cromosomica di P2X7 associata con
la depressione e i disordini bipolari. P2X2 e P2X4 sono inoltre espressi in specifiche
aree cerebrali coinvolte nella depressione, come l’ippocampo, e potrebbero quindi
svolgere un ruolo nella patofisiologia della depressione.
Al fine di ottenere la soppressione dei recettori P2X (P2XR), specifiche
molecole siRNAs sono state disegnate per ogni gene e sono state poi selezionate per
l’attivita’ mediante trasfezione di colture primarie e linee cellulari esprimenti i geni
d’interesse. In questo modo la sequenza siRNA piu’ attiva e specifica per ciascun gene
e’ stata selezionata per la produzione di una cassetta esprimente un “short hairpin RNA”
o shRNA che porta alla produzione del siRNA di interesse all’interno della cellula.
Poiche’ lo scopo finale di questo progetto era il silenziamento a lungo termine dei
recettori P2X nell’ippocampo di ratto, sono stati utilizzati i vettori virali al fine di
garantire l’espressione prolungata del siRNA nei neuroni e nella glia. Infatti i vettori
virali impiegati possono infettare il tessuto cerebrale con elevata efficienza e persistono
episomialmente all’interno della cellula infettata. Per ottenere l’espressione dei siRNAs
nei neuroni, i vettori virali Adeno-associati (AAV) esprimenti shRNA (AAV-shRNA)
sono stati prodotti per ciascun gene bersaglio, poiche’ questi vettori possono infettare i
neuroni con elevata efficienza. Inoltre, vettori Adenovirali 5 (Ad5)-shRNA sono stati
generati per investigare l’effetto della soppressione di P2X7 nelle cellule gliali. Infatti
questi vettori virali infettano preferenzialmente la glia. Per selezionare i vettori piu’
attivi e specifici in grado di sopprimere l’espressione dei recettori P2X in vivo, i vettori
virali sono stati inizialmente testati in vitro in colture primarie. I vettori AAV2/6-
shRNA sono stati validati mediante infezione di neuroni ippocampali. Una riduzione
massima del 80-90% del mRNA dei recettori P2X e’ stata osservata mediante analisi
TaqMan delle cellulare infettate. E’ stato inoltre identificato un vettore Ad5-shRNA in
grado di sopprimere del 80% l’espressione del mRNA per P2X7 in astrociti primari di
ratto. Dati preliminari hanno dimostrato una diminuizione nel profilo funzionale del
recettore P2X7 in cellule microgliali infettate con il vettore Ad5-shRNA. Questi dati
hanno indicato una riduzione statisticamente significativa del recettore bersaglio in
seguito all’infezione con vettori virali esprimenti shRNA in vitro.
Poiche’ le principali evidenze sperimentali, comprendenti sia studi preclinici che
genetici, supportano il coinvolgimento del recettore P2X7 nella pato-fisiologia della
depressione, il lavoro in vivo e’ stato focalizzato su questo gene. Pertanto sia i vettori
virali AAV2/6- che Ad5-shRNA-P2X7 sono stati microinfusi in ippocampo di ratto
utilizzando aghi di vetro. Questa metodica di microiniezione e’ stata selezionata in
quanto in grado di indurre un livello minimo di danno anatomico nel sito di iniezione.
In seguito all’infusione dei vettori virali, la soppressione del gene bersaglio e’ stata
investigata. Nel lavoro in vivo e’ stata dimostrata con successo l’infezione
rispettivamente di neuroni e cellule gliali in seguito all’iniezione nella regione
ippocampale del giro dentato (DG) con i vettori virali AAV2/6- e Ad5-shRNA. Inoltre
una riduzione statisticamente significativa del mRNA per P2X7 e’ stata rilevata
mediante ibridazione in situ nei campioni iniettati con AAV-P2X7-shRNA rispetto al
controllo. Una tendenza simile e’ stata riscontrata nei campioni iniettati con vettore
Ad5-P2X7-shRNA.
In conclusione, in questo studio sono stati generati e validati vettori virali
esprimenti shRNA attivi e specifici, in grado di sopprimere l’espressione del gene
bersaglio in cellule neuronali sia in vitro che in vivo.RNA interference (RNAi) is an evolutionarily conserved process of sequencespecific
post-transcriptional gene silencing that is triggered by double-stranded short
interfering RNA (siRNA). At present RNAi has become an important and widely used
tool for evaluating target gene function both in vitro and in vivo and has been used for
the screening of potential therapeutic targets in pharmaceutical research.
This project has been carried out in order to investigate the applicability of RNA
interference for target gene suppression in the rat central nervous system (CNS) both in
vitro and in vivo. In particular its application in the process of target validation has been
evaluated focusing on the purinergic receptor P2X7, a putative target for mood
disorders and the two related receptors P2X2 and P2X4.
The purinergic receptor P2X7 together with the P2X2 and P2X4 receptors are
members of the ATP-gated cation channel family permeable to sodium, potassium and
calcium. These receptors are expressed in neurons in many regions of the brain where
they play a role in release of neurotransmitters and activation of multiple intracellular
signaling pathways. The P2X7 gene is also expressed in several types of glia where it is
involved in the modulation of neuroinflammatory processes and cytokine release.
In order to obtain P2X receptor (P2XR) suppression, specific siRNAs were
designed for each gene and screened for activity by transfecting both primary cells and
cell lines expressing the target genes. In this way the most active and specific siRNA
sequence for each gene was selected for the production of a short hairpin RNA (shRNA)
cassette leading to intracellular production of the targeting siRNA sequence. Viral
vectors have been used for long term siRNA expression in neurons and glia in the brain
since these vectors can efficiently transduce brain tissue and persist episomally within
the infected cell. In order to drive siRNA expression in neuronal cells, Adeno-associated
viral vectors (AAV) containing an shRNA cassette were produced for each target gene,
since these vectors can infect neurons with high efficiency. Adenovirus 5 (Ad5)-shRNA
vectors targeting P2X7 were also produced to investigate the effects of P2X7
suppression in glia since these viral vectors preferentially infect glia. As a filter to select
the most active and specific viral vectors that could down-regulate P2XRs in vivo, viral
vectors were first tested in vitro in primary cells. The AAV2/6-shRNA vectors were
evaluated by infecting hippocampal neurons. A maximum of 80-90% down-regulation
of P2XR mRNA was observed by TaqMan analysis of the infected cells. An active
Ad5-shRNA vector able to suppress of 80% P2X7 mRNA expression in cultured rat
astrocytes was also identified. Preliminary data demonstrated a statistically significant
decrease in P2X7 function in Ad5-shRNA infected microglial cells. These data indicate
the suppression of target receptor after shRNA viral vector delivery in vitro.
Both the AAV2/6- and Ad5-shRNA-P2X7 viral vectors were then microinfused
into rat hippocampus though glass needles. Transduction of neurons and glia
respectively after infection of the dentate gyrus (DG) region of the hippocampus by
AAV2/6- and Ad5-shRNA vectors was demonstrated. Moreover a statistically
significant down-regulation of P2X7 mRNA in AAV-P2X7-shRNA injected DG
compared to controls was revealed by in situ hybridization. A similar trend was
observed in the Ad5-P2X7-shRNA injected samples.
In conclusion, in this study active and specific shRNA-viral vectors able to
suppress target gene expression in neuronal cells both in vitro and in vivo were
generated and validated
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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