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In Pax8-/- mice the thyroid anlage disappears around E11.5. At birth the gland appears as a rudimentary structure composed only of C cells while thyroid follicular cells are undetectable. Thus, this model does not allow the study of Pax8 function in the postnatal thyroid. To address the role of Pax8 in the differentiated thyroid, I established novel strains amenable to thyroid follicular cell-specific conditional knockout of Pax8 gene using the Cre/lox system. A genetically modified mouse strain (Pax8fl/fl) harbouring a floxed allele of Pax8 was generated. These mice were crossed with two different Cre mice, a Pax8Cre/+ strain expressing constitutively Cre recombinase by embryonic day 8.5 and a Tg-CreER strain in which Cre is active in thyroid follicular cells following tamoxifen injection. Pax8Cre/fl mice do not display an overt phenotype during embryonic life. However, 3-week old Pax8Cre/fl pups show a hypothyroid phenotype characterized by both an increased serum TSH value and a significant reduction in body weight when compared with their wild type littermates. In Pax8Cre/fl animals the thyroid gland is hypoplastic and shows the absence of the follicular structure; moreover, the expression of many thyroid specific genes, required for both hormone production and regulative functions, is completely abolished. In addition, survival of follicular cells is impaired. The disruption of Pax8 in adult life (Pax8ko/fl;Tg-CreER strain) seems to be not efficient and thyroid structure appears comparable to that of wild type. However, the absence of a clear phenotype in the Pax8ko/fl;TgCreER thyroid could be due to the compensatory proliferation of cells in which Pax8 has not been inactivated. In conclusion, in my thesis work I demonstrated in vivo that Pax8 plays a key role in controlling both the expression of many thyroid specific genes and the survival of thyroid follicular cells.

Diatoms are fast-growing and winning competitors in aquatic environments, possibly due to optimized growth performance. However, their life cycles are complex, heteromorphic, and not fully understood. Here, we report on the fine control of cell growth and physiology during the sexual phase of the marine diatom Pseudo-nitzschia multistriata. We found that mating, under nutrient replete conditions, induces a prolonged growth arrest in parental cells. Transcriptomic analyses revealed down-regulation of genes related to major metabolic functions from the early phases of mating. Single-cell photophysiology also pinpointed an inhibition of photosynthesis and storage lipids accumulated in the arrested population, especially in gametes and zygotes. Numerical simulations revealed that growth arrest affects the balance between parental cells and their siblings, possibly favoring the new generation. Thus, in addition to resources availability, life cycle traits contribute to shaping the species ecological niches and must be considered to describe and understand the structure of plankton communities., Description, Growth control of vegetative cells during sexual reproduction favors offspring survival success.

Because of their importance as chemical mediators, the presence of a rich and varied family of lipoxygenase (LOX) products, collectively named oxylipins, has been investigated thoroughly in diatoms, and the involvement of these products in important processes such as bloom regulation has been postulated. Nevertheless, little information is available on the enzymes and pathways operating in these protists. Exploiting transcriptome data, we identified and characterized a LOX gene, PaLOX, in Pseudo-nitzschia arenysensis, a marine diatom known to produce different species of oxylipins by stereo- and regio-selective oxidation of eicosapentaenoic acid (EPA) at C12 and C15. PaLOX RNA interference correlated with a decrease of the lipid-peroxidizing activity and oxylipin synthesis, as well as with a reduction of growth of P. arenysensis. In addition, sequence analysis and structure models of the C-terminal part of the predicted protein closely fitted with the data for established LOXs from other organisms. The presence in the genome of a single LOX gene, whose downregulation impairs both 12- and 15-oxylipins synthesis, together with the in silico 3D protein modelling suggest that PaLOX encodes for a 12/15S-LOX with a dual specificity, and provides additional support to the correlation between cell growth and oxylipin biosynthesis in diatoms.

The Activator Protein-1 transcription factor family (AP-1) transcriptional complex is historically defined as an early response group of transcription factors formed by dimeric complexes of the Jun, Fos, Atf, and Maf bZIP proteins that control cell proliferation and differentiation by regulating gene expression. It has been greatly investigated in many model organisms across metazoan evolution. Nevertheless, its complexity and variability of action made its multiple functions difficult to be defined. Here, we place the foundations for understanding the complexity of AP-1 transcriptional members in tunicates. We investigated the gene members of this family in the ascidian Ciona robusta and identified single copies of Jun, Fos, Atf3, Atf2/7, and Maf bZIP-related factors that could have a role in the formation of the AP-1 complex. We highlight that mesenchyme is a common cellular population where all these factors are expressed during embryonic development, and that, moreover, Fos shows a wider pattern of expression including also notochord and neural cells. By ectopic expression in transgenic embryos of Jun and Fos genes alone or in combination, we investigated the phenotypic alterations induced by these factors and highlighted a degree of functional conservation of the AP-1 complex between Ciona and vertebrates. The lack of gene redundancy and the first pieces of evidence of conserved functions in the control of cell movements and structural organization exerted by these factors open the way for using Ciona as a helpful model system to uncover the multiple potentialities of this highly complex family of bZIP transcription factors.

Ischemic Heart Disease (IHD) remains the developed world's number one killer. The improved survival from Acute Myocardial Infarction (AMI) and the progressive aging of western population brought to an increased incidence of chronic Heart Failure (HF), which assumed epidemic proportions nowadays. Except for heart transplantation, all treatments for HF should be considered palliative because none of the current therapies can reverse myocardial degeneration responsible for HF syndrome. To stop the HF epidemic will ultimately require protocols to reduce the progressive cardiomyocyte (CM) loss and to foster their regeneration. It is now generally accepted that mammalian CMs renew throughout life. However, this endogenous regenerative reservoir is insufficient to repair the extensive damage produced by AMI/IHD while the source and degree of CM turnover remains strongly disputed. Independent groups have convincingly shown that the adult myocardium harbors bona-fide tissue specific cardiac stem cells (CSCs). Unfortunately, recent reports have challenged the identity and the endogenous myogenic capacity of the c-kit expressing CSCs. This has hampered progress and unless this conflict is settled, clinical tests of repair/regenerative protocols are unlikely to provide convincing answers about their clinical potential. Here we review recent data that have eventually clarified the specific phenotypic identity of true multipotent CSCs. These cells when coaxed by embryonic cardiac morphogens undergo a precisely orchestrated myogenic commitment process robustly generating bona-fide functional cardiomyocytes. These data should set the path for the revival of further investigation untangling the regenerative biology of adult CSCs to harness their potential for HF prevention and treatment.

The characterization of multipotent endogenous cardiac stem cells (eCSCs) and the breakthroughs of somatic cell reprogramming to boost cardiomyocyte replacement have fostered the prospect of achieving functional heart repair/regeneration. Areas covered: Allogeneic CSC therapy through its paracrine stimulation of the endogenous resident reparative/regenerative process produces functional meaningful myocardial regeneration in pre-clinical porcine myocardial infarction models and is currently tested in the first-in-man human trial. The in vivo test of somatic reprogramming and cardioregenerative non-coding RNAs revived the interest in gene therapy for myocardial regeneration. The latter, together with the advent of genome editing, has prompted most recent efforts to produce genetically-modified allogeneic CSCs that secrete cardioregenerative factors to optimize effective myocardial repair. Expert opinion: The current war against heart failure epidemics in western countries seeks to find effective treatments to set back the failing hearts prolonging human lifespan. Off-the-shelf allogeneic-genetically-modified CSCs producing regenerative agents are a novel and evolving therapy set to be affordable, safe, effective and available at all times for myocardial regeneration to either prevent or treat heart failure.

The adult mammalian heart, including the human, is unable to regenerate segmental losses after myocardial infarction. This evidence has been widely and repeatedly used up-to-today to suggest that the myocardium, contrary to most adult tissues, lacks an endogenous stem cell population or more specifically a bona-fide cardiomyocyte-generating progenitor cell of biological significance. In the last 15 years, however, the field has slowly evolved from the dogma that no new cardiomyocytes were produced from shortly after birth to the present consensus that new cardiomyocytes are formed throughout lifespan. This endogenous regenerative potential increases after various forms of injury. Nevertheless, the degree/significance and more importantly the origin of adult new cardiomyocytes remains strongly disputed. Evidence from independent laboratories has shown that the adult myocardium harbours bona-fide tissue-specific cardiac stem cells (CSCs). Their transplantation and in situ activation have demonstrated the CSCs regenerative potential and have been used to develop regeneration protocols which in pre-clinical tests have shown to be effective in the prevention and treatment of heart failure. Recent reports purportedly tracking the c-kit+CSC’s fate using Cre/lox recombination in the mouse have challenged the existence and regenerative potential of the CSCs and have raised scepticism about their role in myocardial homeostasis and regeneration. The validity of these reports, however, is controversial because they failed to show that the experimental approach used is capable to both identify and tract the fate of the CSCs. Despite these serious shortcomings, in contraposition to the CSCs, these publications have proposed the proliferation of existing adult fully-matured cardiomyocytes as the relevant mechanism to explain cardiomyocyte renewal in the adult. This review critically ponders the available evidence showing that the adult mammalian heart possesses a definable myocyte-generating progenitor cell of biological significance. This endogenous regenerative potential is expected to provide the bases for novel approaches of myocardial repair in the near future.

Purpose: Multiple myeloma (MM) is a hematologic malignancy characterized by a clonal expansion of plasma cells (PCs) in the bone marrow (BM). Since MM has so far remained incurable, further insights into its pathogenesis and the concomitant identification of new therapeutic targets are urgently needed. The tyrosine kinase receptor EphA3 is known to be involved in various cellular processes including cell viability, cell movement and cell-cell interactions. Recently, EphA3 has emerged as a potential therapeutic target in several hematologic and solid tumors. Here, we aimed to uncover the role of EphA3 in MM. Methods: EphA3 mRNA and protein expression in primary MM bone marrow plasma cells (BMPCs), in MM-derived cell lines and in healthy controls (HCs) was assessed using qRT-PCR, Western blotting and flow cytometry. The effects of siRNA-mediated EphA3 silencing and anti EphA3 antibody (EphA3mAb) treatment on MM PC trafficking and viability were evaluated using in vitro assays. The effects of EphA3mAb treatment were also assessed in two MM-derived mouse xenograft models. Results: We found that EphA3 was overexpressed in primary MM BMPCs and MM-derived cell lines compared to HCs. We also found that siRNA-mediated EphA3 silencing and EphA3mAb treatment significantly inhibited the ability of MM PCs to adhere to fibronectin and stromal cells and to invade in vitro, without affecting cell proliferation and viability. Gene expression profiling showed that EphA3 silencing resulted in expression modulation of several molecules that regulate adhesion, migration and invasion processes. Importantly, we found that EphA3mAb treatment significantly inhibited in vivo tumor growth and angiogenesis in two MM-derived mouse xenograft models. Conclusions: Our findings suggest that EphA3 plays an important role in the pathogenesis of MM and provide support for the notion that its targeting may represent a novel therapeutic opportunity for MM.

Here we describe a conditional doxycycline-dependent mouse model of RET/PTC3 (NCOA4-RET) oncogene-induced thyroid tumorigenesis. In these mice, after 10 days of doxycycline (dox) administration, RET/PTC3 expression induced mitogen activated protein kinase (MAPK) stimulation and a proliferative response which resulted in the formation of hyperplastic thyroid lesions. This was followed, after 2 months, by growth arrest accompanied by typical features of oncogene-induced senescence (OIS), including upregulation of p16INK4A and p21CIP, positivity at the Sudan black B, activation of the DNA damage response (DDR) markers γH2AX and pChk2 T68, and induction of p53 and p19ARF. After 5 months, about half of thyroid lesions escaped OIS and formed tumors that remained dependent on RET/ PTC3 expression. This progression was accompanied by activation of AKT-FOXO1/3a pathway and increased serum TSH levels.

Nucleic acid aptamers have been developed as high-affinity ligands that may act as antagonists of disease-associated proteins. Aptamers are non immunogenic and characterised by high specificity and low toxicity thus representing a valid alternative to antibodies or soluble ligand receptor traps/decoys to target specific cancer cell surface proteins in clinical diagnosis and therapy. The epidermal growth factor receptor (EGFR) has been implicated in the development of a wide range of human cancers including breast, glioma and lung. The observation that its inhibition can interfere with the growth of such tumors has led to the design of new drugs including monoclonal antibodies and tyrosine kinase inhibitors currently used in clinic. However, some of these molecules can result in toxicity and acquired resistance, hence the need to develop novel kinds of EGFR-targeting drugs with high specificity and low toxicity. Here we generated, by a cell-Systematic Evolution of Ligands by EXponential enrichment (SELEX) approach, a nuclease resistant RNA-aptamer that specifically binds to EGFR with a binding constant of 10 nM. When applied to EGFR-expressing cancer cells the aptamer inhibits EGFR-mediated signal pathways causing selective cell death. Furthermore, at low doses it induces apoptosis even of cells that are resistant to the most frequently used EGFR-inhibitors, such as gefitinib and cetuximab, and inhibits tumor growth in a mouse xenograft model of human non-small-cell lung cancer (NSCLC). Interestingly, combined treatment with cetuximab and the aptamer shows clear synergy in inducing apoptosis in vitro and in vivo. In conclusion, we demonstrate that this neutralizing RNA-aptamer is a promising bio-molecule that can be developed as a more effective alternative to the repertoire of already existing EGFR-inhibitors.