The water beetle family Hydraenidae includes more than 1500 species worldwide, currently divided into four subfamilies: Hydraeninae, Ochthebiinae, Prosthetopinae and Orchymontinae. The majority of species are adapted for marginal life in the microscopic, mainly freshwater, aquatic world, feedeing dyatoms algae, even if details about the ecology of these beetles are poorly known. Most Hydraenidae species usually live in a layer of nearly stationary waters, where they adhere to the submerged stones within the fastest regions of cold, clean and fastflowing perennial streams. Hydraena Kugelann represents the largest genus within the water beetle family Hydraenidae, and in particular within Hydraeninae, with about 850 species widely distributed all over the world and several hundreds not yet described. In a recent cladistic analysis, based on morphological characters, Hydraena s.l. was split in two subgenera: Hydraenopsis Janssens (Gondwanian) and Hydraena s.str. (Laurasian). Moreover, within Hydraena s.str, some derived and well-supported monophyletic clades were recognised, and defined as “lineages”. Among them, the "Haenydra" Rey lineage, previously considered by many authors as a valid genus/subgenus, includes about 90 species distributed esclusively in western Palaearctic...
Essendo organismi sessili, le piante hanno evoluto strategie molto sofisticate per la percezione dell’ambiente che le circonda nel loro habitat naturale, allo scopo di adattarvisi con il massimo profitto per la loro sopravvivenza e riproduzione. Le piante ottengono la loro energia dalla radiazione luminosa mediante il processo fotosintetico, e sono quindi particolarmente sensibili alla qualità e quantità della luce che le circonda, percependo in particolar modo la presenza della vegetazione circostante come un segnale di competizione per l’approvvigionamento luminoso. Infatti, crescendo in condizioni di ombreggiatura le piante percepiscono una luce filtrata nella sua componente più fotosinteticamente attiva, caratterizzata da una riduzione nel rapporto tra la radiazione nel rosso (R) e nel rosso lontano (FR) (basso R/FR), a cui reagiscono molto rapidamente aumentando la crescita in allungamento degli organi di sostegno della parte aerea (ipocotile, piccioli) a spese dello sviluppo di foglie e radici (risposta di “fuga dall’ombra”). Se la pianta ha successo nel suo tentativo di crescere al di sopra della vegetazione che la circonda, questa risposta va incontro ad una rapida reversione. D’altra parte, se il segnale persiste la pianta attenua la risposta attraverso l’azione del fattore di trascrizione bHLH HFR1/SICS1. In questo lavoro sono stati integrati studi globali di espressione genica mediante microarray ed analisi genetiche allo scopo di individuare molecole regolative implicate nel controllo della risposta di fuga dall’ombra. I nostri risultati hanno evidenziato che un numero significativo di regolatori trascrizionali sono rapidamente e transientemente indotti dalla luce con basso R/FR (fattori di trascrizione bHLH e HD-Zip). Le analisi condotte hanno anche messo in evidenza un nuovo meccanismo di controllo negativo della risposta di fuga dall’ombra che sembra agire attraverso il fattore di trascrizione bZip HY5. Analisi filogenetiche su alcuni dei regolatori trascrizionali della risposta di fuga dall’ombra (bHLH e HD-Zip II) ci hanno condotto alla loro classificazione in diverse sottofamiglie...
Free disseminated peritoneal tumor cells derive from the detachment from primary cancer and may result in peritoneal carcinomatosis. Peritoneal lavage cytology has low sensitivity in detecting free peritoneal tumor cells; reverse-transcriptase polymerase reaction showed higher sensitivity but low specificity. Our study introduces the combination of the RT-PCR, the immunomagnetic enrichment and the immunofluorescence for the detection of peritoneal free tumor cells.
Materials and Methods
Samples of peritoneal lavage were collected from 22 gastric and 45 colorectal cancer patients; samples were also obtained from 6 patients who underwent abdominal surgery for non-malignant diseases. CEA and CK20 mRNA levels were quantified using a real-time qRT-PCR system. Immunomagnetic enrichment followed by immunofluorescence analysis was performed using monoclonal antibody against the pan-epithelial marker EpCAM/CD326 and polyclonal antibodies against the carcinoembryonic antigen.
For gastric carcinoma the positivity rate for cytology, immunofluorescence and qRT-PCR was 14%, 18% and 77% respectively; for colorectal carcinoma the positivity rate for cytology, immunofluorescence and qRT-PCR was 0%, 18% and 44% respectively. All patients except one when positive at immunofluorescence were also positive at qRT-PCR. All samples of peritoneal lavage from the control group resulted negative for cytology...
New treatments for neuropathic pain
Stefano Cobianchi, Institute of Neuroscience, CNR, Rome (Italy).
Experiment 1: Botulinum neurotoxins.
Aim. Botulinum neurotoxins have been successfully used in clinical practice for the treatment of dystonias and a number of syndromes associated to hyperfunctioning of cholinergic terminals. Recent data support the use of Botulinum neurotoxins (BoNTs) as new therapeutic agents in pain relief. It has been demonstrated that the Botulinum neurotoxin serotype-A is able to induce analgesia in inflammatory pain conditions (1). In this study we investigated the effects of two different serotypes, A and B (BoNT/A and BoNT/B), in the development and recovering from neuropathic pain in mice subjected to the sciatic nerve ligation (Chronic Constriction Injury, Bennett and Xie model) (2, 3).
Methods. Mice were subcutaneously injected into the plantar surface of both hindpaws either with BoNT/A (two doses: 7.5 or 15 pg/paw), BoNT/B (3.75 pg/paw) or saline, on different days before and after CCI. The temporal trend of neuropathy over a long time interval (80 days) was analyzed measuring the mechanical allodynic response to the Dynamic Aesthesiometer Test. Functional recovery of the injured paw was followed examining the mice walking pattern...
In human endothelial cells, nitric oxide (NO) results in class IIa histone deacetylases (HDACs) activation and
marked histone deacetylation. It is unknown whether similar epigenetic events occur in embryonic stem cells (ESC) exposed to NO and how this treatment could influence ESC therapeutic potential during tissue regeneration. This study reports that the NO-dependent class IIa HDACs subcellular localization and activity decreases the global acetylation level of H3 histones in ESC and that this phenomenon is associated with the inhibition of Oct4, Nanog, and KLF4 expression. Further, a NO-induced formation of macromolecular complexes including HDAC3, 4, 7, and protein phosphatase 2A (PP2A) have been detected. These processes correlated with the expression of the mesodermalspecific protein brachyury (Bry) and the appearance of several vascular and skeletal muscle differentiation markers. These events were abolished by the class IIa-specific inhibitor MC1568 and by HDAC4 or HDAC7 short interfering RNA (siRNA). The ability of NO to induce mesodermic/cardiovascular gene expression prompted us to evaluate the regenerative potential of these cells in a mouse model of hindlimb ischemia. We found that NO-treated ESCs injected into the cardiac left ventricle selectively localized in the ischemic hindlimb and contributed to the regeneration of muscular and vascular structures. These findings establish a key role for NO and class IIa HDACs modulation in ESC mesodermal commitment and enhanced regenerative potential in vivo.
Natural killer (NK) cells play a critical role in host defense against viral infections.
Chronic HIV-1 infection is associated with an accumulation of dysfunctional NK
cell, that unsuccessfully control viral replication. However, the underlying
mechanisms for this NK cell dysfunction are poorly understood.
NK cells do not express an antigen
specific receptor, as do other lymphocytes. Instead, NK cells encode a variety of
different activating and inhibitory receptors and NK cell activation is dependent on a
delicate balance between signals of opposite sign elicited by the multiple NK cell
receptor–ligand interactions that follow NK cell–target cell interaction.
The goal of my PhD project was to analyze the role of NK cell activating pathways
upon HIV-1 infection.
Specifically, I studied the modulation of the activating receptor NKG2D with
particular interest on the mechanism of release, namely shedding, of its ligands
(NKG2DLs) during HIV-1 infection in in vitro cell systems as well as in HIVinfected
Moreover, I investigated the modulation by viral proteins, such as Nef and Vpu, of
PVR (Poliovirus Receptor, CD155, Necl-5) a ligand for another stimulatory
receptor, DNAM-1 (DNAX accessory molecule-1 or CD226).The final goal of this work was to identify novel factors that regulate the function of
immune responses against HIV-1 and that could provide new prognostic biomarkers
and innovative antiviral strategies.; The work was supported by grants of the Italian Ministry of Health...
La leucemia mieloide acuta (LAM) rappresenta un gruppo eterogeneo di disordini ematopoietici, caratterizzati da distinte lesioni genetiche. La leucemia promielocitica acuta (LAP) è un peculiare sottotipo di LAM in cui l’arresto della maturazione mieloide si verifica allo stadio di promielocita, ed è causata dall’oncoproteina di fusione PML/RARα che interferisce con il differenziamento mieloide reprimendo la trascrizione dei geni responsivi all’acido retinoico tutto–trans (ATRA). Il gruppo in cui ho svolto la mia attività di ricerca ha precedentemente dimostrato che un ristretto gruppo di microRNA (miRNA), piccoli RNA non codificanti che regolano l’espressione genica a livello post-trascrizionale, è differenzialmente espresso in blasti di LAP ed è modulato dal trattamento differenziante con ATRA. Il confronto dell’espressione di questi miRNA in promielociti normali e in blasti di LAP alla diagnosi, ha rivelato in questi ultimi una ridotta espressione del miRNA let-7c. Il let-7c appartiene alla famiglia di miRNA let-7, che gioca ruoli importanti in varie attività cellulari, e diversi membri di questa famiglia sono specificamente repressi in vari tipi di tumori. Per quanto riguarda le leucemie, il let-7c è ipo-espresso anche in pazienti affetti da LAM con t(8;21) e inv16.
Sulla base di questi dati...
hMENA, a member of the Ena/VASP proteins involved in the cytoskeletal regulation during cell motility and adhesion, is up-regulated in invasive tumor cells and plays a crucial role in metastatic progression. We show that hMENA alternative splicing is related to EMT process and this is in line with recent data demonstrating that hMENA belongs to a cluster of genes whose alternative splicing is related to EMT.
Our group has previously characterized two hMENA isoforms, one 88-kDa isoform defined as “classic” and one splice variants, named hMENA11a (90 kDa). Here we report a novel splice variant of human MENA, named hMENAΔv6 (80 kDa). hMENA11a characterizes epithelial cancer cell lines expressing E-Cadherin, whereas hMENAΔv6 is expressed in breast and cervix tumor cell lines displaying the phenotypic features of EMT with migratory behaviour. The analysis of pancreatic cell lines indicates that hMENA11a is expressed in the normal human pancreatic ductal epithelial cell line (HPDE) besides epithelial pancreatic tumor cell lines (CFPAC, T3M4, and PACA44), whereas hMENAΔv6 is expressed in PANC1 cells that lack hMENA11a and express Vimentin.
We observed that the Ser157 of VASP is phosphorylated either in normal or pancreatic cancer cell lines with an epithelial phenotype and expressing hMENA11a...
Megalencephalic leukoencephalopathy with subcortical cysts (MLC), is a rare congenital and incurable leukodystrophy characterized by macrocephaly, subcortical fluid cysts and myelin vacuolation. The majority of MLC patients carry mutations in the MLC1 gene encoding a membrane protein named MLC1 that is highly expressed in brain astrocytes contacting blood vessels, ependyma and meninges. Although the neuropathological features of MLC disease, the molecular structure and the cellular localization of MLC1 suggest a possible involvement of this protein in astrocyte-mediated osmoregulatory processes, the function of MLC1 is still unknown. Understanding the function of MLC1 protein whose mutations are the main cause of MLC is an essential step toward identification of disease mechanisms and development of effective therapies.
During the course of this thesis project we generated new data on MLC1 expression, distribution and functional associated pathways in astrocytes that are deregulated by pathological mutations, paving the way for the identification of the specific MLC1 function. We found that: i) endogenous MLC1 protein is expressed in cultured astrocytes, particularly in the plasma membrane where it interacts with caveolin-1 and proteins of the dystrophin/dystroglycan complex (DCG)...
Exon Skipping has been demonstrated to be a successful strategy for the gene therapy of Duchenne Muscular Dystrophy (DMD): the rational is to convert severe Duchenne forms into milder Becker ones. Here we show the selection of U1 snRNA-antisense constructs able to confer effective rescue of dystrophin synthesis in a Δ44 Duchenne genetic background, through skipping of exon 45; moreover, we demonstrate that the resulting dystrophin is able to recover the correct timing of myogenic marker expression, to re-localize nNOS and to rescue expression of miRNAs previously shown to be sensitive to the Dystrophin-nNOS-HDAC2 pathway.
Becker mutations display different phenotypes, likely depending on whether the shorter protein is able to reconstitute the wide range of wild type functions. Among them, efficient assembly of the dystrophin associated protein complex (DAPC) and Nitric Oxide Synthase (nNOS) localization are important. Comparing different Becker deletions we demonstrate the correlation between the ability of the mutant dystrophin to re-localize nNOS and the expression levels of two miRNAs, miR-1 and miR29c, known to be involved in muscle homeostasis and to be controlled by the Dys-nNOS-HDAC2 pathway.
Since the gene responsible for the disease has been identified...
Duchenne Muscular Dystrophy (DMD), caused by mutations in the dystrophin gene, is one of the most severe myopathies. Among different therapeutic strategies, exon skipping allows the rescue of dystrophin synthesis through the production of a shorter but functional mRNA. Making use of exon skipping strategy we demonstrated that in DMD, the absence of dystrophin at the sarcolemma delocalizes and downregulates Nitric Oxide Synthase (nNOS); this alters HDAC2 S-nitrosylation and its chromatin association. We show that the differential HDAC2 nitrosylation state in Duchenne versus wild-type conditions deregulates the expression of a specific subset of microRNA genes crucial in DMD physiopathology.
Namely, we identified miR-1 as regulator of the redox state of the cell through modulation of the G6PD enzyme while miR-29 controls the fibrotic process targeting extracellular matrix proteins.
We also show that, at variance with other myomiRs, miR-206 and miR-31 escape from the dystrophin-nNOS control being expressed in activated satellite cells before dystrophin expression. In these cells, miR-206 contributes to muscle regeneration through repression of the satellite specific factor Pax7, while miR-31 inhibits the early expression of dystrophin by directly repressing its mRNA.
Recent advances in DNA sequencing have changed the field of genomics as well as that of proteomics making it possible to generate gigabases of genome and transcriptome sequence data at substantially lower cost than it was possible just ten years ago. In recent years, many high-throughput technologies have been developed to interrogate various aspects of cellular processes, including sequence and structural variation and the transcriptome, epigenome, proteome and interactome.
These Next Generation Sequencing (NGS) experimental technologies are more mature and accessible than the computational tools available
for individual researchers to move, store, analyse and present data in a user-friendly and reproducible fashion. My research work is placed in this scenario and focuses on the analysis of data produced by NGS technologies as well as on the development of new tools aimed at solving the different problems that arise during NGS data analysis. In order to achieve this aim, my group and I have dealt with several open biomedical problems in collaboration with different research groups of the Sapienza University. Some of these experiments have already given interesting results but mostly have represented the occasion and starting point for the development of new tools able to improve some crucial steps of the analyses...