http://elea.unisa.it/xmlui/handle/10556/88 2026-04-20T12:00:29Z 2026-04-20T12:00:29Z Ruggiero, Paola http://elea.unisa.it/xmlui/handle/10556/2040 2025-04-30T14:23:11Z 2015-07-16T00:00:00Z

Analisi funzionale del gene UVR8 e suo ruolo nella risposta delle piante a stress ambientali Ruggiero, Paola Plants are sessile organisms and, therefore, are continuously subjected to environmental sub-optimal or stressful conditions. In an arid environment plants are challenging multiple stresses, such, as water shortage, excessive soil salinity, osmotic stress conditions and high light intensity, including an excess of ultraviolet light mainly (UV-B). To overcome these unfavorable conditions, plants have evolved different strategies to adapt to common osmotic stress and high UV-B light. Recently, the UV-B photoreceptor, UVR8 (UV RESISTANCE LOCUS 8), has been identified and its role in the plant response to UV-B largely clarified. Besides its role in UV-B signaling, we have demonstrated that the expression of UVR8 gene is strongly induced by osmotic and salt stress in wild type A. thaliana seedling (Fasano et al., 2014). Moreover, by using a "gain and loss of function" approach we have evidenced a role of the UVR8 gene in plant growth, development and differentiation: UVR8 overexpressing plants have a reduced vegetative growth (minor diameter of the rosette, smaller leaves, height less), while silenced plants are characterized by a higher growth and produce a large number of siliques and seeds (Fasano et al., 2009; 2010), reminiscent of the response SIMR (Stress Induced Morphogenic Response). The UVR8 protein is predominantly localized in the cytoplasm and in response to low UV-B doses only a small fraction monomerizes and translocates to the nucleus, where it acts as a transcriptional activator. Most of the UVR8 protein remains in the cytoplasmic proteins and it might exert additional cellular functions by interacting with other proteins involved in the complex plant response to environmental stresses. This project was aimed at the identification of putative proteins that interact with UVR8 protein, and to establish a functional role of these interactions in plant responses to osmotic stress. The main results are summarized below: 1. by using complementary approaches of proteomics and immunoprecipitation, several potential proteins that interact with the UVR8 protein were identified; in particular, our attention was focused on the proteins APX1 (Ascorbate peroxidase) and GGT1 (glutamate-glyoxylate-aminotransferase), known for their role in the mechanisms of detoxification of H2O2, a reactive oxygen species that accumulates in the plant cell in response to different environmental conditions that generate an oxidative stress; 2. the interaction between APX1-UVR8 and UVR8-GGT1 were confirmed in vivo, by using two different assays: the BiFC and the co-immunoprecipitation; 3. through a functional analysis, it was shown that different levels of the UVR8 protein are associated with a different level of ROS, in response to conditions of osmotic stress, suggesting a possible function associated to the interaction of these between UVR8 e APX1 4. a gene expression analysis of the stress marker gene RD29 and the gene GGT1 in UVR8- knock-out or overexpressing plants was performed, in response to salt stress. These experiments provided an early indication of the effect of different levels of the UVR8 protein on the transcriptional level of these two genes and, more generally, in the global response to salt stress in Arabidopsis plants. Further analyses are required to establish whether the interaction of UVR8 with APX1 or GGT1 might somehow influence their enzymatic activity. In addition, previous studies have shown that UVR8 binds to COP1 (an E3-ubiquitin ligase) and targets negative regulators of the UV-B dependent pathway to proteasome degradation (Huang X et al., 2013). The use of inhibitors of this proteoliytic pathway may contribute to determine whether UVR8 protein can recruit APX or GGT1 proteins in order to stabilize them or target them to the proteolytic degradation in response to direct or osmotic stress derived oxidative stress. 2010 - 2011

2015-07-16T00:00:00Z Sannino, Anna http://elea.unisa.it/xmlui/handle/10556/1356 2025-04-30T14:16:06Z 2013-02-06T00:00:00Z

Valutazione dell’induzione di risposta adattativa a mutageni in colture cellulari di mammifero in seguito ad esposizioni a campi elettromagnetici non ionizzanti Sannino, Anna Nell’ambito del progetto di ricerca dal titolo “Valutazione dell’induzione di risposta adattativa a mutageni in colture cellulari di mammifero in seguito ad esposizioni a campi elettromagnetici non ionizzanti” è stato caratterizzato l’effetto protettivo (risposta adattativa) dell’esposizione a radiofrequenza (RF) dal danno indotto in colture cellulari da agenti a nota azione genotossica. La sperimentazione è stata eseguita in colture cellulari primarie (linfociti umani da 27 donatori sani) e in colture stabilizzate di roditore (fibroblasti di polmone di criceto, V79). In una fase iniziale, l’attività di ricerca ha riguardato l’approfondimento di osservazioni precedenti, dove si era riscontrato che linfociti umani da sangue periferico, pre-esposti ad un campo elettromagnetico alla frequenza di 900 MHz, segnale GSM, e trattati con Mitomicina C (MMC) mostravano un danno cromosomico ridotto rispetto ai trattamenti con sola MMC (Sannino et al., 2009). Applicando il test del micronucleo (MN) col blocco della citodieresi, è stato infatti, dimostrato che la pre-esposizione a RF è in grado di proteggere dal danno al DNA solo quando viene effettuata nella fase S del ciclo cellulare, inducendo risposta adattativa (RA), ma non ha alcun effetto in fase G0 o G1. Successivamente, l’attenzione è stata focalizzata su un segnale di telefonia mobile di terza generazione quale il segnale UMTS alla frequenza di 1950 MHz per valutare il ruolo a) dei parametri dell’esposizione (frequenza, modulazione e tasso di assorbimento specifico, SAR), b) del mutageno impiegato e c) del modello cellulare nella RA indotta da RF. La sperimentazione su colture cellulari di linfociti da sangue periferico ha evidenziato che anche pre-esposizioni a 1950 MHz sono in grado di evocare risposta adattativa, ma il grado di protezione dal danno indotto da MMC è strettamente dipendente dal SAR applicato. Inoltre, le stesse condizioni di esposizione si sono mostrate efficaci anche nella protezione di danno cromosomico indotto da trattamenti con raggi X, evidenziando che pre-esposizioni a RF sono in grado di ridurre il danno al DNA indipendentemente dalla natura e dal meccanismo di azione del mutageno impiegato. Infatti, mentre la MMC è un agente alchilante che induce cross-link nella molecola di DNA, i raggi X sono un agente clastogeno che induce rotture del singolo e doppio filamento. Risultati analoghi sono stati ottenuti quando sono state impiegate le V79 come modello cellulare, mostrando che il fenomeno dell’adattamento da RF non è limitato a cellule primarie quali i linfociti umani ma si riscontra anche in linee cellulari stabilizzate, sebbene in quest’ultimo caso siano richieste condizioni sperimentali più spinte, sia in termini di pre-trattamento (SAR) che di dosi di MMC. L’ultima parte del lavoro sperimentale ha riguardato la valutazione dei possibili meccanismi di azione alla base della RA indotta da RF, sulla base delle indicazioni riportate in letteratura sulla RA indotta da radiazioni ionizzanti. A tale scopo, nelle condizioni sperimentali che davano adattamento nei due tipi cellulari studiati, sono stati valutati effetti sulla vitalità cellulare (test di esclusione del tripan blue), sulla progressione del ciclo cellulare (test citofluorimetrico di incorporazione dello ioduro di propidio) e sul sistema di riparo del DNA (inibizione degli enzimi di riparazione mediante trattamento con 3-Aminobenzamide, 3AB). Nel caso dei linfociti umani è stata anche valutata l’apoptosi mediante il test citofluorimetrico dell’annessina V-FITC/ioduro di propidio. I risultati ottenuti sia con i linfociti umani sia con le V79 indicano che i meccanismi di azione alla base della risposta adattativa indotta da RF non coinvolgono la vitalità, il ciclo cellulare e l’apoptosi. Invece, è stato evidenziato un possibile ruolo degli enzimi di riparo del DNA nell’induzione del fenomeno. Infatti, in colture trattate con 3AB, che inibisce il legame della poli(ADP-ribosio) polimerasi alla cromatina, non si osserva adattamento. Riassunto E’ interessante sottolineare che, a conferma delle osservazioni riportate in questo progetto di ricerca, nel periodo di svolgimento del presente dottorato altri gruppi hanno riscontrato la capacità della RF ad indurre RA sia in vitro in colture di HL-60 (Jin et al., 2012) sia in vivo in topi e ratti (Cao et al., 2010, 2011; Jiang et al., 2012; Mortazavi et al., 2011, 2012) valutando differenti target biologici. [a cura dell'autore] 2010 - 2011

2013-02-06T00:00:00Z Carratù, Anna http://elea.unisa.it/xmlui/handle/10556/1324 2025-04-30T14:15:24Z 2013-02-13T00:00:00Z

Le complicanze cardiovascolari nel diabete mellito: studio delle basi molecolari Carratù, Anna Diabetes is characterized by development of specific microvascular complications and by a high incidence of accelerated atherosclerosis. The assumption underlying current clinical treatment is that lowering the level of time-averaged glucose concentration, measured as hemoglobin A1c (HbA1c), prevents the development and progression of microvascular complications. This current treatment recommendation, adopted by diabetes professional societies around the world, is based on data from the 1993 Diabetes Control and Complications Trial (DCCT). Recent Diabetes Control and Complications Trial data analyses show that 89% of the variation in microvascular complications risk in type 1 diabetes is not captured by HbA1c values (time-averaged glucose concentration). Recent experimental evidence from Dr Brownlee’s lab, shows that transient exposure to threshold levels of high glucose reprograms human endothelial cells to continue overproducing reactive oxygen species in the presence of physiologic glucose concentrations. This persistent ROS overproduction causes an equally persistent overexpression of pro-inflammatory genes in normal glucose due to hystone modifications in the proximal promoter of the NF-κB subunit p65. Since in normal cells the epigenetic changes are rapidly reversed by histone demethylases and histone methyltransferases my thesis work aimed to understand how transient exposure to high glucose reprograms endothelial cells, and characterize the critical regulatory networks that shift vascular endothelial cells to a state of persistent excess ROS production after transient exposure to high glucose. We found that transient spikes of hyperglycemia cause persistent mitochondrial overproduction of ROS during subsequent periods of prolonged normal glucose, causing persistent activation of the epigenetic changes and resultant vascular inflammatory gene expression. We identified a multi-component positive feedback loop induced by transient exposure to high glucose in human vascular endothelial cells which maintains persistently increased ROS production in normal glucose, thus we verified that transient disruption of any of the elements in the feed back loop rapidly restores the system to its normal state, including reversing persistent increased ROS production, persistent hyperglycemia-induced epigenetic changes and persistent increased NF-κB-dependent pro-inflammatory gene expression. Our results highlight the dramatic and long-lasting effects that short-term hyperglycemic spikes can have on vascular cells and suggest that transient spikes of hyperglycemia may be an HbA1c–independent risk factor for diabetic complications. Moreover we understood the mechanism underlying the continue overproducing of reactive oxygen species in the presence of physiologic glucose concentrations in human endothelial cells. This knowledge will provide the basis for developing new type 1 diabetes treatment paradigms that more effectively prevent the development and progression of microvascular complications. [edited by author] 2010 - 2011

2013-02-13T00:00:00Z Montefusco, Sandro http://elea.unisa.it/xmlui/handle/10556/1311 2025-04-30T14:17:00Z 2012-01-26T00:00:00Z

Studio del ruolo funzionale e caratterizzazione strutturale del cuprocomplesso TFF1-Cu Montefusco, Sandro The maintenance of gastrointestinal tissue integrity is physiologically essential in the presence of the persistent harassment of microbial flora and injurious agents. Even though the repair of the gastric epithelium may be modulated by several factors, the epithelial continuity also depends on a family of small peptides called trefoil factors (TFFs). The trefoil factors family comprises the gastric peptides pS2/TFF1, the spasmolytic peptide (SP)/TFF2 and the intestinal trefoil factor (ITF)/TFF3; they are characterized by a three looped domain, the “trefoil domain”, stabilized by three disulphide bridges. TFF1 and TFF3 also have a seventh cysteine that allows the formation of omo- and/or hetero-dimers. On the other hand TFF2 presents only a monomeric form, containing two trefoil domains in the same polypeptide chain. TFFs are small protease-resistant proteins that are abundantly produced by mucus-secreting cells of the gastrointestinal tract onto the mucosal surface. TFFs are essential in the protection of the mucosal epithelia against a wide range of biological threats, thus contributing to the mucosal repair. The signaling events that mediate the cellular responses elicited by TFFs are only partially understood. Moreover there are convincing evidence that TFFs do play an important role in tumorigenesis, even though their specific roles in cancer are still unclear. TFF1 expression is strongly induced after mucosal injury and it has been proposed that TFF1 functions as a gastric tumor suppressor gene. Several studies confirm that TFF1 expression is frequently lost in gastric cancer because of deletions, mutations or methylation of the TFF1 gene. Infection by Helicobacter pylori, a class 1 carcinogen according to WHO classification, is thought to promote stomach carcinogenesis through induction of aberrant DNA methylation. Samples from infected patients show lower expression of TFF1. Recent studies have also shown that there is a direct relationship between Helicobacter pylori and the dimeric form of the protein. In fact, it was demonstrated that the core oligosaccharide portion of H. pylori lipopolysaccharide (RF-LPS) is able to bind to TFF1. It also seems that the loss of TFF1 is an important event in shaping the NF-kB-mediated inflammatory response during the progression to gastric tumorigenesis, being TFF1 a negative regulator of NF-kB signalling. It is thus emerging a clear correlation between loss of TFF1, the development of inflammatory disease and the neoplastic process. Recent analyses made by our research group allowed us to point out the up-regulation of TFF1 gene expression in rats fed on copper deficient diets, and allowed us to find out the unexpected ability of TFF1 to bind copper ions. The presence of a cysteine surrounded by several negatively charged residues in the carboxy-terminus of the protein suggested the presence of a copper-binding site. Afterwards, it was shown that Cys58 and at least three Glu surrounding residues are essential to efficiently bind copper. Moreover, the incubation of the native peptide with copper salts increases the fraction of peptide omodimers produced by inter-molecular oxidation of Cys58 and disulphide bond formation. The Ph.D. research project was aimed at characterising the structure-function relationship of the TFF1-Cu complex. Briefly, we studied the influence of copper on known TFF1 biological activities and on its gene regulation, then we investigated its involvement in the TFF1 mediated mechanisms of Helicobacter pylori virulence and infection. A preliminary Real Time PCR quantitative analysis showed that copper deficiency positively modulates tff1 expression in an adenocarcinoma cell line (AGS), thus confirming our previous data obtained in vivo in copper deficient rat intestine. In order to map possible copper responsive elements in the proximal promoter sequence, we analysed the expression of a reporter gene (Luciferase) driven by deletion constructs of the tff1 gene promoter. AGS cells transfected with the deletion constructs allowed us to identify the upstream 5’ gene sequence -583/-435 as a promoter region sensitive to the changes of copper concentration. In fact, copper chelation treatments with bathocuproine disulfonate (BCS) were able to stimulate an increase of the promoter activity of the corresponding deletion construct. Following the sequence analysis (Transfac software) we focused our attention on a putative SP1 binding site identified in this region, whose binding ability was then confirmed by electrophoretic mobility shift assay (-561/-552). In agreemente with our in vitro results, it was also observed that copper favours the native TFF1 dimer formation in the culture medium of MCF-7 and HT29-MTX cells (a mucus-secreting clone obtained from the HT29 colon cancer cell line), thus confirming a possible role of the metal in the balance between the monomeric and the dimeric forms To evaluate the effect of copper-TFF1 interaction on the well known motogenic activity of the protein, we performed wound healing assays on an inducible clone of gastric cancer cells (AGS) able to overexpress the peptide (AGS-AC1). As expected, the overexpression of TFF1 stimulates an appreciable increase of cell migration, and copper chelation (BCS) undo the benefits of the increased peptide level. Our previous results showed that copper treatments decreased the amount of secreted protein in culture medium. Further experiments demonstrated that induced AGS-AC1 cells are able to store intracellularly higher amount of copper if compared to uninduced AGS-AC1 cells. This evidence suggests that TFF1 levels may also play a role also in the uptake/traffic of copper in this in vitro model. Finally, we studied the combined influence of TFF1 and copper in Helicobacter pylori infections. Our results demonstrate that Cu-TFF1 complex promotes H. pylori colonization of AGS cells. In fact, AGS-AC1 cells overexpressing TFF1 are more efficiently colonised by H. pylori wild-type (str. P12) if compared to uninduced cells. The presence of copper in a duplicate experiment further increases the colonization, as well as copper chelation by bathocuproine disulfonate (BCS) reduces the observed effect. The same result was obtained with H. pylori str. P12Δ479, an isogenic mutant expressing a truncated LPS core still able to bind to TFF1. On the other hand, H. pylori P12Δ1191, unable to bind TFF1, is not affected by copper levels in the culture medium. Parallel experiments were carried out on mucus secreting HT29-E12 goblet cells, to compare and/or confirm the results obtained in AGS-AC1. The results show that also in HT29-E12 cells the H. pylori colonization follows a similar trend, increasing when incubated in the presence of Cu and decreasing after BCS treatment. The present work contributed interesting results in the field of the biochemistry of the epithelia, in the wake of the research in progress in our laboratory aimed at studying the biological activities of the newly identified metalloprotein Cu-TFF1, whose properties are still poorly characterized. On the basis of the previous structural pieces of evidence we observed that the protein level and the balance of its oligomeric forms can be affected and regulated by copper ions. In turn, this delicate equilibrium is able to affect the integrity and the rheological properties of the epithelial barrier, thus representing a fine tuner, or an Achille’s heel, through which pathogenic microrganisms and deregulated proliferation of neoplastic cells may take advantage for their invasiveness. The role of copper in the TFFs biochemistry represents a new finding in the puzzling and versatile functions of this interesting peptide family, whose thorough comprehension still reserves many questions and surprises. [edited by author] 2009 - 2010

2012-01-26T00:00:00Z