Ruolo dei fattori di trascrizione Pbx1 e Pbx2 nella morfogenesi degli arti

Lo sviluppo degli arti dei vertebrati avvien lungo tre assi: prossimo-distale (PD), antero-posteriore (AP) e dorso-ventrale. La specificazione di tali assi e’ regolata da centri organizzativi quali la ZPA (zona ad attivita’ polarizzante) e l’AER (cresta apicale ectodermica). In particolare lo sviluppo prossimo-distale necessita della formazione di un feedback loop tra Shh, espresso nella ZPA, e l’AER. La famiglia dei Pbx include quattro geni: Pbx1, Pbx2, Pbx3 e Pbx4 (Selleri et al. 2001; Selleri et al. 2004; Rhee et al. 2004; Monica et al. 1991; Popperl et al. 2000).E’ stato dimostrato che Pbx1 e’ necessario durante l’organogenesi e lo sviluppo dello scheletro sia assiale che appendicolare (Selleri et al, 2001). In particolare topi knockout per Pbx1 mostrano difetti morfologici a carico dei cingoli scapolari e pelvici, dello sviluppo dello stilopodio sia anteriore che posteriore (omero e femore), mentre la regione distale degli arti (zugopodio ed autopodio) non sembra essere affetta. In questa tesi dimostriamo che Pbx2 e’ espresso, durante le prime fas della morfogenesi degli arti, in tutto il mesoderma, Tale risultato suggerisce la presenza di un “Pbx code” lungo gli assi AP e PD con funzioni Pbx1/ Pbx2 sovrapposte e complementari. Tale evidenza e’ ulteriormente avvalorata dal fenotipo ottenuto negli embrioni doppi mutanti Pbx1-/-;Pbx2-/- in cui si ha la completa assenza di arti sia anteriori che posteriori. Il fenotipo invece caratterizzante gli embrioni Pbx1-/-;Pbx2+/- fornisce un valido modello per studiare i meccanismi alle base dello sviluppo degli arti. Tali embrioni, infatti, mostrano difetti scheletrici sia assiali che appendicolari molto piu’ severi di quelli riscontrati negli embrioni Pbx1-/- nonchè una letalità, precoce (E13.5) rispetto ai Pbx1-/- , ma più avanzata rispetto ai doppi mutanti omozigoti (E10.5), permettendone quindi uno studio piu’ approfondito. Tale studio indica che nei vertebrati, a differenza di quanto avviene in Drosophila, lo sviluppo distale degli arti e’ Pbx1/Pbx2-dipendente. Il ruolo dei Pbx in tale processo e’ mediato , almeno a livello degli arti posteriori, dal controllo gerarchico che, tali proteine, esercitano sulla distrubuzione dei trascritti dei geni Hox e di Shh. In generale, i dati ottenuti durante il periodo del dottorato, dimostrano che, a livello degli arti posteriori, attraverso il controllo della distribuzione degli Hox e la regolazione della ZPA, Pbx1/Pbx2 esercitano una funzione gerarchica sui geni Hox piuttosto che fungere esclusivamente da loro cofattori

Epsins function in Notch signaling activation

In mechanistic terms, endocytosis is the process by which plasma membrane (PM) components, together with extracellular solutes, macromolecules and particles, are internalized in the cell. Once the endocytic vesicle (or vacuole) is formed by fission of the PM, it is generally delivered to a specialized membrane compartment – the endosome – for recycling, degradation or re-routing. In cell-physiological terms, endocytosis exerts multiple functions, which are only partially known and characterized. At a minimum, it maintains PM homeostasis by counterbalancing the apposition of new membrane (due to exocytosis) and by renewing PM components. More extensively, endocytosis constantly modulates PM composition and takes an active part in a variety of normal and pathological cell processes, including cell nutrition, cell motility, mitosis, neurotransmission, immune response, and microorganism entry. In recent years, much of the effort to investigate this extensive endocytic activity has been focused upon unveiling the reciprocal interplay between endocytosis and cell signaling. Our laboratory, in collaboration with the laboratory of Pietro De Camilli (Yale University, USA) has pioneered the use of genetic models in mice to study several aspects of the endocytic function, mostly at the synapse. Recently, we generated mice models for a highly conserved gene family of multidomain adaptors – the epsin family - whose function was linked to endocytosis. By characterizing the phenotypic defects of the epsin1/2 double knockout mice, we found that epsins are essential components of the machinery required for Notch signaling activation during embryogenesis in mammals [1]. More recently, we characterized that epsins molecular action is exerted in a ubiquitin- dependent endocytic reaction that triggers the internalization of the Notch ligand, a process necessary for the activation of the Notch receptor. Our preliminary data extend epsin function to Notch signaling activation in primary keratinocytes and to VEGF signaling modulation in angiogenesis

Histological findings of osteonecrosis spotted prior to tooth extractions. Should we consider tooth extraction still the main trigger event?

Osteonecrosis of the jaw (ONJ) is an adverse drug reaction described as the progressive destruction and death of bone that affects the mandible and maxilla of patients exposed to the treatment with medications known to increase the risk of disease, in the absence of a previous radiation treatment. Tooth extraction often precedes the manifestation of ONJ; indeed, it is sometimes called trigger event and it have also been considered as risk factors for the onset of ONJ. As a consequence, some of the guidelines recommend avoiding tooth extractions in patients at risk of ONJ; however, a growing body of evidence indicates that dental/periodontal infection prior to extraction, rather than dental extraction may represent the main local risk factor for ONJ. Ten patients at risk of ONJ have undergone tooth extractions. They were identified and included in our retrospective monocentric clinical investigation. Patients underwent tooth extractions with standardized procedures (PROMaF protocol), and bone biopsies were taken. Extractions were performed due to symptomatic, non-restorable teeth in patient at risk of ONJ; histological findings of ONJ were observed in all samples. This outcome may highlight that the proof of non‐exposed ONJ might be the histopathologic confirmation of necrotic bone, as stated by European task force on MRONJ. Additionally, alveolar biopsy should possibly be taken in every case of suspected ONJ, which needs an proper and prompt management for successful healin

Effects of Cutting Patterns of Shears on Occlusion Processes in Pruning of High-Quality Wood Plantations

Arboriculture plantations aim to produce high-quality wood. In order to investigate the type and extent of mechanical injury that pruning causes to tree cambium as well as the effects on the healing process, different types of shear were selected and used in an eight-year-old Quercus robur L. plantation. The amount of removed, detached and crushed bark was assessed by means of image analysis immediately after pruning. After 15 months, the effect of different cutting patterns on the healing process was investigated by measuring the area of the pruned branch covered by woundwood (HI1). Five years after pruning, the same analysis was performed above and below bark (HIo5 and HIu5) and a number of parameters were assessed in order to quantify the quantity and quality (symmetry) of woundwood growth and the healing time for sealing. The action of pruning tools depends on cutting pattern and branch diameter. The greater the diameter, the longer the healing time. The double-blade tool caused less injury and showed the fastest healing process. The use of double blade pruning tools is thus recommended to improve the performance of wood quality production in arboriculture plantations. We also recommend the healing index HI1 for an early assessment of pruning damage

Plasmodium falciparum liver stage antigen-1 is cross-linked by tissue transglutaminase

Background Plasmodium falciparum sporozoites injected by mosquitoes into the blood rapidly enter liver hepatocytes and undergo pre-erythrocytic developmental schizogony forming tens of thousands of merozoites per hepatocyte. Shortly after hepatocyte invasion, the parasite starts to produce Liver Stage Antigen-1 (LSA-1), which accumulates within the parasitophorous vacuole surrounding the mass of developing merozoites. The LSA-1 protein has been described as a flocculent mass, but its role in parasite development has not been determined. Methods Recombinant N-terminal, C-terminal or a construct containing both the N- and C- terminal regions flanking two 17 amino acid residue central repeat sequences (LSA-NRC) were subjected to in vitro modification by tissue transglutaminase-2 (TG2) to determine if cross-linking occurred. In addition, tissue sections of P. falciparum-infected human hepatocytes were probed with monoclonal antibodies to the isopeptide ε-(γ-glutamyl)lysine cross-bridge formed by TG2 enzymatic activity to determine if these antibodies co-localized with antibodies to LSA-1 in the growing liver schizonts. Results This study identified a substrate motif for (TG2) and a putative casein kinase 2 phosphorylation site within the central repeat region of LSA-1. The function of TG2 is the post-translational modification of proteins by the formation of a unique isopeptide ε-(γ-glutamyl)lysine cross-bridge between glutamine and lysine residues. When recombinant LSA-1 protein was crosslinked in vitro by purified TG2 in a calcium dependent reaction, a flocculent mass of protein was formed that was highly resistant to degradation. The cross-linking was not detectably affected by phosphorylation with plasmodial CK2 in vitro. Monoclonal antibodies specific to the very unique TG2 catalyzed ε- lysine cross-bridge co-localized with antibodies to LSA-1 in infected human hepatocytes providing visual evidence that LSA-1 was cross-linked in vivo. Conclusions While the role of LSA-1 is still unknown these results suggest that it becomes highly cross-linked which may aid in the protection of the parasite as it develo

Mouse Models for Epsin Function

The vertebrate skin is a barrier-forming organ in which keratinocytes form a highly organized, stratified epithelium protecting the organism from the outside environment. One of the major regulators of this structure is Notch signaling. Notch is a transmembrane receptor interacting with ligands expressed on the surface of neighboring keratinocytes. Keratinocyte-specific deletion of Notch signaling pathway impairs epidermal differentiation, resulting in skin-barrier defects and skin carcinogenesis. Our laboratory, by a genetic approach in mice, demonstrates that combined inactivation of Epsin1 and Epsin2 genes leads to embryonic lethality around E9.5-10. The phenotype of Epn1;Epn2 double knockout is characterized by a subversion of the three main developmental programs active at this developmental stage, i.e., cardiovascular development, somitogenesis, and neural tube differentiation. Collectively, these morphological alterations resemble the developmental defects observed in mutants of Notch genes or in genes essential for the activation of the Notch signaling pathway, suggesting a crucial role of Epsin in enabling Notch signaling during embryogenesis. Intriguingly, the apparently healthy Epn1+/-;Epn2-/- shows an high incidence of squamous papillomas on their skin. Furthermore, expression of another epsin family member originally localized exclusively to surface epithelia, Epsin3, dramatically increases in the hyperplastic lesions of the three-allele mutants and in human basal carcinomas. In order to get further insight on Epn3 function we performed morphological expression analyses during mouse development. In contrast with initial reports, both in embryos and adults, we could detect various levels of Epn3 expression in several tissues, i.e., surface epithelia, neural tissue and heart. Moreover, in vitro studies performed on human keratinocytes in culture show a prominent role of this Epsin in the regulation of Notch signaling in this cell compartment