Impiego di un vettore lentivirale basato sul virus dell'immunodeficienza umana di tipo 1 per il trasferimento genico di siRNA.

Questo lavoro di tesi si è focalizzato sull’impiego di un vettore lentivirale basato sul Virus dell’Immunodeficienza Umana di tipo 1 (HIV-1) per introdurre un siRNA specifico per il silenziamento del Vascular Endothelial Growth Factor (VEGF) in cellule di epitelio pigmentato retinico umano (ARPE-19).ope

G9a co-suppresses LINE1 elements in spermatogonia

BACKGROUND: Repression of retrotransposons is essential for genome integrity and the development of germ cells. Among retrotransposons, the establishment of CpG DNA methylation and epigenetic silencing of LINE1 (L1) elements and the intracisternal A particle (IAP) endogenous retrovirus (ERV) is dependent upon the piRNA pathway during embryonic germ cell reprogramming. Furthermore, the Piwi protein Mili, guided by piRNAs, cleaves expressed L1 transcripts to post-transcriptionally enforce L1 silencing in meiotic cells. The loss of both DNA methylation and the Mili piRNA pathway does not affect L1 silencing in the mitotic spermatogonia where histone H3 lysine 9 dimethylation (H3K9me2) is postulated to co-repress these elements. RESULTS: Here we show that the histone H3 lysine 9 dimethyltransferase G9a co-suppresses L1 elements in spermatogonia. In the absence of both a functional piRNA pathway and L1 DNA methylation, G9a is both essential and sufficient to silence L1 elements. In contrast, H3K9me2 alone is insufficient to maintain IAP silencing in spermatogonia. The loss of all three repressive mechanisms has a major impact on spermatogonial populations inclusive of spermatogonial stem cells, with the loss of all germ cells observed in a high portion of seminiferous tubules. CONCLUSIONS: Our study identifies G9a-mediated H3K9me2 as a novel and important L1 repressive mechanism in the germ line. We also demonstrate fundamental differences in the requirements for the maintenance of L1 and IAP silencing during adult spermatogenesis, where H3K9me2 is sufficient to maintain L1 but not IAP silencing. Finally, we demonstrate that repression of retrotransposon activation in spermatogonia is important for the survival of this population and testicular homeostasis

A transit amplifying population underpins the efficient regenerative capacity of the testis

The spermatogonial stem cell (SSC) that supports spermatogenesis throughout adult life resides within the GFRα1-expressing A type undifferentiated spermatogonia. The decision to commit to spermatogenic differentiation coincides with the loss of GFRα1 and reciprocal gain of Ngn3 (Neurog3) expression. Through the analysis of the piRNA factor Miwi2 (Piwil4), we identify a novel population of Ngn3-expressing spermatogonia that are essential for efficient testicular regeneration after injury. Depletion of Miwi2-expressing cells results in a transient impact on testicular homeostasis, with this population behaving strictly as transit amplifying cells under homeostatic conditions. However, upon injury, Miwi2-expressing cells are essential for the efficient regenerative capacity of the testis, and also display facultative stem activity in transplantation assays. In summary, the mouse testis has adopted a regenerative strategy to expand stem cell activity by incorporating a transit-amplifying population to the effective stem cell pool, thus ensuring rapid and efficient tissue repair

Oligoasthenoteratozoospermia and Infertility in Mice Deficient for miR-34b/c and miR-449 Loci

Male fertility requires the continuous production of high quality motile spermatozoa in abundance. Alterations in all three metrics cause oligoasthenoteratozoospermia, the leading cause of human sub/infertility. Post-mitotic spermatogenesis inclusive of several meiotic stages and spermiogenesis (terminal spermatozoa differentiation) are transcriptionally inert, indicating the potential importance for the post-transcriptional microRNA (miRNA) gene-silencing pathway therein. We found the expression of miRNA generating enzyme Dicer within spermatogenesis peaks in meiosis with critical functions in spermatogenesis. In an expression screen we identified two miRNA loci of the miR-34 family (miR-34b/c and miR-449) that are specifically and highly expressed in post-mitotic male germ cells. A reduction in several miRNAs inclusive of miR-34b/c in spermatozoa has been causally associated with reduced fertility in humans. We found that deletion of both miR34b/c and miR-449 loci resulted in oligoasthenoteratozoospermia in mice. MiR-34bc/449-deficiency impairs both meiosis and the final stages of spermatozoa maturation. Analysis of miR-34bc-/-;449-/- pachytene spermatocytes revealed a small cohort of genes deregulated that were highly enriched for miR-34 family target genes. Our results identify the miR-34 family as the first functionally important miRNAs for spermatogenesis whose deregulation is causal to oligoasthenoteratozoospermia and infertility

In vitro effect of retinoic acid and epigenetic modifying drugs on mesenchymal stem cells

Effetto in vitro dell'acido retinoico e dei farmaci epigenetici sulle cellule staminali mesenchimal

Impiego di un vettore lentivirale basato sul virus dell'immunodeficienza umana di tipo 1 per il trasferimento genico di siRNA.

Questo lavoro di tesi si è focalizzato sull’impiego di un vettore lentivirale basato sul Virus dell’Immunodeficienza Umana di tipo 1 (HIV-1) per introdurre un siRNA specifico per il silenziamento del Vascular Endothelial Growth Factor (VEGF) in cellule di epitelio pigmentato retinico umano (ARPE-19)

miR-34bc/449 regulates a small cohort of genes in spermatocytes.

<p>(<b>A</b>) Expression scatterplot showing relative average expression of affymetrix probes between control (x-axis) and miR-34bc^−/−;449^−/− (y-axis). Significantly deregulated (p = 0.05) genes with a log₂ fold change of 0.5 (red) are shown. (<b>B</b>) The list of the 13 upregulated genes with predicted miR-34 seed binding sites is shown. Also indicated is the gene function as well as number of miR-34 binding sites. (<b>C</b>) qRT-PCR expression analysis of representative miR-34 family seed-containing deregulated genes identified. Normalized data are plotted as relative fold change in miR-34bc^−/−;449^−/− versus wild type pachytene spermatocytes. Standard error is shown and the asterisk indicates significantly upregulated expression (P<0.05). Other genes identified from the array that change in expression are also presented. The data in all panels are from biological quadruplicates of each genotype. (<b>D</b>) Sylamer enrichment landscape plot for all 876 7 nt words complementary to canonical mouse miRNA seed regions. The y-axis represents the sorted genelist of 21,560 genes from most up-regulated to most down-regulated in the miR-34bc^−/−;449^−/− pachytene spermatocytes. Each 7mer word was tested for significant enrichment across the 3′UTRs of genes in this list. The word corresponding to seed matching miR-34 family (Red) is enriched in the up-regulated genes.</p