Modellizzazione del tasso di assorbimento idrico e nutritivo della gerbera (Gerbera jamesonii H. Bolus, cv. Vital) coltivata in idroponica

L’elemento tecnico più importante nella coltivazione fuori suolo è l’alimentazione idrica e nutritiva della coltura effettuata attraverso la fertirrigazione, cioè la somministrazione congiunta di acqua e sali minerali. Lo studio delle relazioni tra le condizioni ambientali e la crescita, l’assorbimento idrico e nutritivo della coltura può consentire lo sviluppo di modelli matematici da implementare in software di gestione, anche relativamente semplici, o di simulazione da utilizzare, ad esempio, in fase di pianificazione produttiva a livello aziendale o addirittura di comprensorio produttivo. Lo studio condotto per la tesi ha avuto come obiettivo principale la modellizzazione dei consumi di acqua e di elementi nutritivi di una coltura su substrato (lana di roccia) a ciclo chiuso di gerbera, condotta simulando la disponibilità di acqua irrigua di diversa qualità (alta o bassa salinità). La scelta di questa specie è stata motivata dalla sua importanza commerciale e dalla sua relativa sensibilità allo stress salino. Più in particolare, per l’assorbimento minerale sono stati considerati diversi modelli, di tipo meccanicistico oppure empirico-statistici (modelli di regressione), con il tasso di crescita e di consumo di acqua tra le variabili indipendenti. Per l’assorbimento idrico, invece, è stato calibrato e validato il modello di traspirazione proposto da Baille et al. (1994), che è una semplificazione del modello fisico di Penman-Monteith e che è stato testato in numerose specie (cetriolo, Medrano et al., 2005; zucchino, Rouphael e Colla G., 2004; rosa, Kittas et al., 1999; Suay et al., 2003; pomodoro, Boulard e Jeema., 1993), compresa la gerbera, oggetto di uno studio di Marfà et al. (2000) pur meno dettagliato di quello riportato in questa tesi. Se soddisfacenti sono stati i risultati relativi alla calibrazione e validazione del modello della traspirazione, mentre i modelli di nutrizione minerale, di varia natura, hanno dimostrato una minore capacità di simulare (quindi, prevedere) il tasso (settimanale) di assorbimento di elementi minerali. Il fatto che, dal punto di vista statistico, i risultati migliori siano stati ottenuti considerando, in un modello di regressione, il tasso di crescita e di assorbimento idrico (peraltro, positivamente correlati tra di loro), suggerisce un’ipotesi per un ulteriore esperimento: verificare se quest’ultimo influenza la nutrizione minerale attraverso un genuino effetto fisiologico, legato al flusso di massa a livello radicale, oppure attraverso un effetto indiretto, cioè condizionando il rifornimento minerale, come in effetti è stato in questo studio, in cui il consumo idrico della coltura era automaticamente compensato aggiungendo soluzione nutritiva completa al sistema idroponico

The Intracellular DNA Sensor IFI16 Gene Acts as Restriction Factor for Human Cytomegalovirus Replication

Human interferon (IFN)-inducible IFI16 protein, an innate immune sensor of intracellular DNA, modulates various cell functions, however, its role in regulating virus growth remains unresolved. Here, we adopt two approaches to investigate whether IFI16 exerts pro- and/or anti-viral actions. First, the IFI16 gene was silenced using specific small interfering RNAs (siRNA) in human embryo lung fibroblasts (HELF) and replication of DNA and RNA viruses evaluated. IFI16-knockdown resulted in enhanced replication of Herpesviruses, in particular, Human Cytomegalovirus (HCMV). Consistent with this, HELF transduction with a dominant negative form of IFI16 lacking the PYRIN domain (PYD) enhanced the replication of HCMV. Second, HCMV replication was compared between HELFs overexpressing either the IFI16 gene or the LacZ gene. IFI16 overexpression decreased both virus yield and viral DNA copy number. Early and late, but not immediate-early, mRNAs and proteins were strongly down-regulated, thus IFI16 may exert its antiviral effect by impairing viral DNA synthesis. Constructs with the luciferase reporter gene driven by deleted or site-specific mutated forms of the HCMV DNA polymerase (UL54) promoter demonstrated that the inverted repeat element 1 (IR-1), located between −54 and −43 relative to the transcription start site, is the target of IFI16 suppression. Indeed, electrophoretic mobility shift assays and chromatin immunoprecipitation demonstrated that suppression of the UL54 promoter is mediated by IFI16-induced blocking of Sp1-like factors. Consistent with these results, deletion of the putative Sp1 responsive element from the HCMV UL44 promoter also relieved IFI16 suppression. Together, these data implicate IFI16 as a novel restriction factor against HCMV replication and provide new insight into the physiological functions of the IFN-inducible gene IFI16 as a viral restriction factor

The Elk-1 and Serum Response Factor Binding Sites in the Major Immediate-Early Promoter of Human Cytomegalovirus Are Required for Efficient Viral Replication in Quiescent Cells and Compensate for Inactivation of the NF-κB Sites in Proliferating Cells▿

The major immediate-early promoter (MIEP) region of human cytomegalovirus (HCMV) plays a critical role in the regulation of lytic and latent infections by integrating multiple signals supplied by the infecting virus, the type and physiological state of the host cell, and its extracellular surroundings. The interaction of cellular transcription factors with their cognate binding sites, which are present at high densities within the enhancer upstream from the MIEP core promoter, regulate the rate of IE gene transcription and thus affect the outcome of HCMV infection. We have shown previously that the NF-κB binding sites within the MIEP enhancer and cellular NF-κB activity induced by HCMV infection are required for efficient MIEP activity and viral replication in quiescent cells (P. Caposio, A. Luganini, G. Hahn, S. Landolfo, and G. Gribaudo, Cell. Microbiol. 9:2040-2054, 2007). We now show that the inactivation of either the Elk-1 or serum response factor (SRF) binding site within the enhancer also reduces MIEP activation and viral replication of recombinant HCMV viruses in quiescent fibroblasts. In these cells, we show that the expression of either Elk-1 or SRF is required for optimal IE gene expression, and that the HCMV-stimulated activation of the MEK1/2-ERK1/2 signaling axis leads to Elk-1 transcriptional competency. Furthermore, the replication kinetics of recombinant viruses in which NF-κB, Elk-1, and SRF binding sites all are inactivated demonstrate that the higher levels of Elk-1 and SRF binding to MIEP in proliferating cells can compensate even for a lack of HCMV-induced NF-κB-mediated MIEP transactivation. These observations highlight the importance of the combination of different MIEP binding sites to optimize IE gene expression in cells in different physiological states

The US16 gene of human cytomegalovirus is equired for efficient viral infection of endothelial and epithelial cells.[Bronzini M.*, Luganini A.* co-first author]

The human cytomegalovirus (HCMV) US12 gene family comprises a set of 10 contiguous genes (US12 to US21), each encoding a predicted seven-transmembrane protein and whose specific functions have yet to be ascertained. While inactivation of individual US12 family members in laboratory strains of HCMV has not been found to affect viral replication in fibroblasts, inactivation of US16 was reported to increase replication in microvascular endothelial cells. Here, we investigate the properties of US16 further by ascertaining the expression pattern of its product. A recombinant HCMV encoding a tagged version of the US16 protein expressed a 33-kDa polypeptide that accumulated with late kinetics in the cytoplasmic virion assembly compartment. To elucidate the function(s) of pUS16, we generated US16-deficient mutants in the TR clinical strain of HCMV. According to previous studies, inactivation of US16 had no effect on viral replication in fibroblasts. In contrast, the US16-deficient viruses exhibited a major growth defect in both microvascular endothelial cells and retinal pigment epithelial cells. The expression of representative IE, E, and L viral proteins was impaired in endothelial cells infected with a US16 mutant virus, suggesting a defect in the replication cycle that occurs prior to IE gene expression. This defect must be due to an inefficient entry and/or postentry event, since pp65 and viral DNA did not move to the nucleus in US16 mutant-infected cells. Taken together, these data indicate that the US16 gene encodes a novel virus tropism factor that regulates, in a cell-specific manner, a pre-immediate-early phase of the HCMV replication cycle

Eosinophilic esophagitis: clinical, endoscopic, histologic and therapeutic differences and similarities between children and adults

In the absence of secondary causes, eosinophilic esophagitis (EoE) is a chronic, local, progressive, T-helper type 2 immune-mediated disorder characterized by symptoms of esophageal dysfunction and eosinophil-predominant inflammation. In the last 20 years, the incidence and prevalence of EoE have risen sharply, and the chances of encountering affected patients in clinics and endoscopy rooms have increased. Nevertheless, it is estimated that the mean diagnostic delay of EoE is 4-6 years in both children and adults. Unfortunately, the longer the disease stays unrecognized, the likelier it is for the patient to have persistent or increased esophageal eosinophilic inflammation, to complain of non-resolving symptoms, and to develop fibrotic complications. Early detection depends on the recognition of initial clinical manifestations that vary from childhood to adulthood and even among patients of the same age. The disease phenotype also influences therapeutic approaches that include drugs, dietary interventions, and esophageal dilation. We have herein reviewed epidemiologic, clinical, endoscopic, and histologic features and therapeutic options of EoE focusing on differences and similarities between children and adults that may certainly serve in daily clinical practice

Suppression of HCMV replication by IFI16 does not require IFN-β antiviral activity.

<p>HELFs were electroporated with a mixture of four different small interfering RNA (siRNA IFN-β) or scrambled control siRNA (siRNA ctrl), or left not electroporated (NE), and then infected with AdV IFI16 or AdV LacZ (MOI of 200 PFU/cell) before subsequent infection with HCMV 24 hours later (MOI of 0.1 PFU/cell). A) Total RNA was isolated 24 hours post HCMV infection and IFN-β mRNA expression was determined by quantitative real-time PCR. Levels of IFN-β mRNA are presented normalized to the levels of cellular β-actin. Experiments were repeated at least three times and one representative result is shown (mean ± SD). B) Cell-free supernatants were harvested at the indicated hours post infection (hpi) and virus amounts determined by plaque assay. The data shown are the average of three experiments ± SD (*p<0.05, **p<0.01, one-way ANOVA followed by Bonferroni's post test).</p

Interplay between Sp1 and IFI16 down-regulates HCMV replication.

<p>A) Nuclear cell protein extracts from HELFs mock infected (lanes 1, 4, 7), infected with HCMV at an MOI of 2 for 24 hours (lanes 2, 5, 8), or infected with AdV IFI16 or AdV LacZ (data not shown) at an MOI of 200 for 24 hours and then with HCMV (lanes 3, 6, 9) were immunoprecipitated with polyclonal antibodies against IFI16 or control antibody (ctrl). Samples were then immunoblotted with polyclonal antibodies against Sp1 or IFI16. B) Infected cell lysates were immunoprecipitated with anti-IFI16 polyclonal antibodies in the absence (lane 2 or 4) or presence of benzonase (lanes 3 and 5) or with control antibody (ctrl). Proteins from the IP were analyzed by Western blotting using polyclonal antibody recognizing Sp1. C) Nuclear extracts from HELFs mock infected or infected with AdV IFI16 or AdV LacZ at an MOI of 200 for 24 hours and then with HCMV at an MOI of 2 for 24 hours were analyzed by ChIP assay using anti-Sp1 rabbit polyclonal antibody. Sp1-coprecipitating DNA was analyzed by quantitative real-time PCR with IR-1 sequence specific primers. An unrelated rabbit polyclonal anti-serum was used as control (data not shown). Non-immunoprecipitated whole cell extract (input) obtained from AdV LacZ- or AdV IFI16-infected cells was employed to normalize the IR-1 viral DNA subjected to immunoprecipitation. Experiments were repeated at least three times and one representative result is shown (mean ± SD) (***p<0.001, unpaired t test). D) Nuclear cell protein extracts from HELFs stably transduced with the recombinant Lentivirus carrying the full-length IFI16 protein (IFI16wt), or dnIFI16 ORFs (ΔDIFI16 or ΔBIFI16), were infected with HCMV at an MOI of 2 for 24 hours, immunoprecipitated with monoclonal antibodies against flag V5 or control antibodies (ctrl), and immunoblotted with polyclonal antibodies against Sp1 (top panel) or flag V5 (bottom panel).</p

IFI16 protein is a negative regulator of Herpesvirus replication.

<p>A) HELFs were electroporated with a mixture of four different small interfering RNAs (siRNA IFI16), scrambled control siRNA (siRNA ctrl), or left not electroporated (NE), and then infected with the indicated viruses at a multiplicity of 0.05 PFU/cell. Cell-free supernatants were harvested on the indicated hours post infection (hpi) and virus amounts determined by plaque assays. The data shown are the average of three experiments ± SD (*p<0.05, **p<0.01, ***p<0.001, one-way ANOVA followed by Bonferroni's post test). B) HELFs were transfected with siRNA IFI16 or siRNA ctrl or left not electroporated (NE) and IFI16 expression assayed by Western blotting on the indicated days (d) with anti-IFI16 polyclonal Abs. β-actin was included as a loading control. C) HELFs were treated with siRNA as described for panel A and then infected with HCMV at an MOI of 0.05 (left panel) or 1 (right panel) PFU/cell. Cell-free supernatants were harvested on the indicated hours post infection and the virus amounts determined by plaque assays. The data shown are the average of three experiments ± SD.</p