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Giorgia PAVESI

Personale tecnico amministrativo
Dipartimento di Scienze della Vita sede ex-Scienze Farmaceutiche

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Pubblicazioni

2021 - Intrinsic Fluorescence of the Active and the Inactive Functional Forms of Human Thymidylate Synthase [Articolo su rivista]
Vitiello, Simone; Caselli, Monica; Pavesi, Giorgia; Santucci, Matteo; Ferrari, Stefania; Costi, Maria Paola; Ponterini, Glauco
abstract

The observables associated with protein intrinsic fluorescence – spectra, time decays, anisotropies – offer opportunities to monitor in real time and non-invasively a protein‘s functional form and its interchange with other forms with different functions. We employed these observables to sketch the fluorometric profiles of two functional forms of human thymidylate synthase (hTS), a homodimeric enzyme crucial for cell proliferation and thus targeted by anticancer drugs. The protein takes an active and an inactive form. Stabilization of the latter by peptides that, unlike classical hTS inhibitors, bind it at the monomer/monomer interface offers an alternative inhibition mechanism that promises to avoid the onset of drug resistance in anticancer therapy. The fluorescence features depicted herein can be used as tools to identify and quantify each of the two protein forms in solution, thus making it possible to investigate the kinetic and thermodynamic aspects of the active/inactive conformational interchange. Two examples of fluorometrically monitored interconversion kinetics are provided.

2019 - Excited-state intramolecular proton transfer in a bioactive flavonoid provides fluorescence observables for recognizing its engagement with target proteins [Articolo su rivista]
Vanossi, D.; Caselli, M.; Pavesi, G.; Borsari, C.; Linciano, P.; Costi, M. P.; Ponterini, G.
abstract

A benzothiophene-substituted chromenone with promising activity against Leishmania and Trypanosoma species exhibits peculiar fluorescence properties useful for identifying its complexes with target proteins in the microorganism proteomes. The emission spectra, anisotropy and time profiles of this flavonoid strongly change when moving from the free to the protein-bound forms. The same two types of emission are observed in organic solvents and their mixtures with water, with the relative band intensities depending on the solvent ability to establish hydrogen bonds with the solute. The regular emission prevails in protic solvents, while in aprotic solvents the anomalously red-shifted emission occurs from a zwitterionic tautomeric form, produced in the excited state by proton transfer within the intramolecularly H-bonded form. This interpretation finds support from an experimental and theoretical investigation of the conformational preferences of this compound in the ground and lowest excited state, with a focus on the relative twisting about the chromenone-benzothiophene interconnecting bond. An analysis of the absorption and emission spectra and of the photophysical properties of the two emitting tautomers highlights the relevance of the local microenvironment, particularly of the intra- and intermolecular hydrogen bonds in which this bioactive compound is involved, in determining both its steady-state and time-resolved fluorescence behaviour.

2016 - Intracellular quantitative detection of human thymidylate synthase engagement with an unconventional inhibitor using tetracysteine-diarsenical-probe technology [Articolo su rivista]
Ponterini, Glauco; Martello, Andrea; Pavesi, Giorgia; Lauriola, Angela; Luciani, Rosaria; Santucci, Matteo; Pela', Michela; Gozzi, Gaia; Pacifico, Salvatore; Guerrini, Remo; Marverti, Gaetano; Costi, Maria Paola; D'Arca, Domenico
abstract

Demonstrating a candidate drug' s interaction with its target protein in live cells is of pivotal relevance to the successful outcome of the drug discovery process. Although thymidylate synthase (hTS) is an important anticancer target protein, the efficacy of the few anti-hTS drugs currently used in clinical practice is limited by the development of resistance. Hence, there is an intense search for new, unconventional anti-hTS drugs; there are approximately 1600 ongoing clinical trials involving hTS-targeting drugs, both alone and in combination protocols. We recently discovered new, unconventional peptidic inhibitors of hTS that are active against cancer cells and do not result in the overexpression of hTS, which is a known molecular source of resistance. Here, we propose an adaptation of the recently proposed tetracysteine-arsenic-binding-motif technology to detect and quantitatively characterize the engagement of hTS with one such peptidic inhibitor in cell lysates. This new model can be developed into a test for high-throughput screening studies of intracellular target-protein/small-molecule binding.

2016 - TRPA1 Is Expressed in Central But Not in Peripheral Glia [Articolo su rivista]
Vellani, Vittorio; Gomis-Perez, Carolina; Pinti, Marcello; Prandini, Massimiliano; Pavesi, Giorgia; Giacomoni, Chiara; Caprini, Marco
abstract

TRPA1 are cation channels expressed in sensory neurons and in several other cell types. This channel is specifically activated by ally isothiocyanate (AITC), the pungent component of mustard oil, as well as by other electrophilic compounds. Although TRPA1 expression in central glia has been reported, its subcellular localization and its expression in peripheral glia have not been investigated before. In this paper we report the molecular and functional expression of TRPA1 in rat cortical astrocytes. Real-time RT-PCR identified low but significant amounts of TRPA1 mRNA in cortical astrocytes while no signal was seen in peripheral glia isolated from dorsal root ganglia (DRG) or in a glial cell line (DITNC-1). Calcium imaging showed AITC-induced signals in astro-cytes while no response in peripheral glia. AITC induced calcium signals in astrocytes in the presence and in the absence of extracellular calcium, suggesting an intracellular localization of TRPA1 channels. Whole cell electrophysiological recordings were performed in astrocytes, in peripheral glia and in DITNC-1 cells transfected with TRPA1 during AITC application. In TRPA1-transfected DITNC-1 cells typical TRPA1 currents were recorded with a reversal potential near 0 mV, consistent with the opening of a non-selective cation channel. No such currents were recorded in untransfected DITNC-1 cells, in astrocytes and in peripheral glial cells, where even high concentrations of AITC (up to 10 mM) induced no significant outward current. In astrocytes AITC transiently induced an outward rectifying current with the reversal potential near ?90 mV, consistent with K channel activation, likely activated by intracellular release of calcium. Our results suggest that TRPA1 channels are molecularly and functionally expressed in calcium-containing organelles of rat cortical astrocytes, with no expression in the plasma membrane.

2014 - Internalization and stability of a thymidylate synthase peptide inhibitor in ovarian cancer cells [Articolo su rivista]
Cannazza, Giuseppe; Cazzato, ADDOLORATA STEFANIA; Marraccini, Chiara; Pavesi, Giorgia; Pirondi, Silvia; R., Guerrini; M., Pelà; Frassineti, Chiara; Ferrari, Stefania; Marverti, Gaetano; Ponterini, Glauco; Costi, Maria Paola
abstract

Information on the cellular internalization and stability of the ovarian cancer cell growth inhibitor peptide, LSCQLYQR (LR), is vital for lead optimization. Ad-hoc-synthesized LR/fluorescent-probe conjugates were used to monitor the internalization of the peptide. Mass spectrometry was used to identify adducts resulting from the thiol reactivity of the cysteine residue in LR. A mechanistic model is proposed to explain the observed change in intracellular peptide amount over time. Structural modifications can be foreseen to improve the peptide stability.

2013 - A proteomic approach to investigate the mechanism of action of anticancer peptides [Relazione in Atti di Convegno]
Genovese, Filippo; Gualandi, Alessandra; Taddia, Laura; Caselli, Monica; Ponterini, Glauco; Ferrari, Stefania; Marverti, Gaetano; R., Guerrini; M., Pela'; Pavesi, Giorgia; C., Trapella; Costi, Maria Paola
abstract

Many efforts to improve survival of patients affected by Ovarian Cancer (OC) have focused on more effective systemic therapies and on the search for new therapeutic targets. One of the molecular targets for OC is human Thymidylate Synthase (hTS), a homodimeric enzyme essential for DNA biosynthesis. The main goal of our research is to identify compounds able to inhibit hTS by interfering with its dimerization, without causing its over-expression and the onset of cellular drug resistance against the traditional hTStargeted compounds. We have recently discovered some peptides which specifically target the hTS dimer interface and inhibit the enzyme by stabilizing its di-inactive form [1]. These molecules have been recently investigated for their SAR profile. LR, our lead compound, inhibits the intracellular enzyme in both cisplatin (cDDP)-sensitive and -resistant ovarian cancer cells without causing protein overexpression, thus showing a potential for overcoming the limits of OC chemotherapy. This work aims at setting up a proteomic approach able to provide information on the changes in the protein expression profile induced in OC cells by treatment with LR with respect to a well-known folate antimetabolite, Pemetrexed (PTX) and identify key proteins that are involved in its mechanism of action.

2013 - Invited lecture to 18th World Congress on Advances in Oncology and 16th International Symposium on Molecular Medicine 10-12 October, 2013, Creta Maris, Hersonissos, Crete, Greece [Relazione in Atti di Convegno]
Genovese, Filippo; Gualandi, Alessandra; Marverti, Gaetano; Taddia, Laura; Glauco, Ponterini; Pirondi, Silvia; Pela', Michela; Pavesi, Giorgia; Costi, Maria Paola
abstract

Proteomic approach to the identification of early phase biomarker for anticancer peptides targeting thefolate pathway. F.Genovesea, A.Gualandia,b L.Taddiaa, G.Ponterinia, G.Marvertia, S.Pirondia, R.Guerrinic, M.Pelàa,c G.Pavesia, C.Trapellac, M.P.Costia aUniversity of Modena and Reggio Emilia, via Campi 183, 41125 Modena, Italy, bCRBA, S. Orsola University Hospital, Bologna, Italy, cDepartment of Pharmaceutical Science, University of Ferrara, Italy Many efforts to improve survival of patients affected by Ovarian Cancer (OC) have focused on more effective systemic therapies and on the search for new therapeutic targets. One of the molecular targets for OC is human Thymidylate Synthase (hTS), a homodimeric enzyme essential for DNA biosynthesis. In order to investigate the effects of hTS-interface-mimicking peptides at a cellular level, we started a study in which the cellular behavior of the peptides was investigated in combination with the proteomic differential analysis of the cytoplasmatic proteins of treated vs. untreated OC cells. The same experiment was performed with pemetrexed (PTX), a well known antifolate, for control purposes. The bioinformatic analysis of the effects of our peptide drug candidate indicates that deregulations can be mainly assigned to modulation of translational initiation, termination of RNA Pol-II transcription, transport, and protein catabolic events. Although apparently folate pathway members are not directly altered at a protein level, as the selection of ions to be sequenced is stochastic and biased towards abundant peptides, the bioinformatic analysis of peptide-modulated proteins suggested cellular investigations on the proteins of the folate-associated genes showing the largest number of dependencies to the species of the core set, which is required for the phosphorylation of several deoxyribonucleosides and nucleoside analogues. Comparison with the PTX-modulated proteins shows that some proteins of the proteasome complex and ribonucleoproteins are involved in both cases. These differences suggest that the two compounds may show a different mechanism of action which is in agreement with the hypothesized pharmacological model. Detailed cellular proteins profile based on the inferred roles of the identified proteins will further clarify the biological effects. 1.A proteomic approach to investigate the mechanism of action of anticancer peptides. F. Genovese, A. Gualandi, L. Taddia, M. Caselli, G. Ponterini, S.Ferrari, G. Marverti, R. Guerrini, M. Pela, G. Pavesi, C. Trapella, M.P. Costi, Proceeding 32EPS, p.466, ISBN 978-960-466-121-3). This work is supported by AIRC-DROC IG10474. www.unimore.airc-droc.it

2012 - Proteomic Approach to the Detection of the Mechanism of Action of Anticancer Peptides [Abstract in Rivista]
Genovese, Filippo; Gualandi, Alessandra; Taddia, Laura; Caselli, Monica; Ponterini, Glauco; Marverti, Gaetano; Pirondi, Silvia; R., Guerrini; M., Pela'; Pavesi, Giorgia; C., Trapella; Costi, Maria Paola
abstract

A label-free quantitative proteomic approach has been undertaken to study the effects of the peptide on the proteins involved in the modulated metabolic pathways, in particular those involved in the folate metabolism. Structure-activity relationships (SAR) have been performed to improve the lead peptide pharmacodynamics. All the compounds have been assayed and a protein profile set was studied to mark and validate their behavior as inhibitor of OC cell growth.

2011 - Functional endothelin receptors are selectively expressed in isolectin B4-negative sensory neurons and are upregulated in isolectin B4-positive neurons by neurturin and glia-derived neurotropic factor. [Articolo su rivista]
Vellani, Vittorio; Prandini, Massimiliano; C., Giacomoni; Pavesi, Giorgia; Ravegnani, Laura; Magherini, Pier Cosimo
abstract

Activation of endothelin receptors expressed in DRG neurons is functionally coupled to translocation of PKCε from cytoplasm to the plasma membrane. Using immunocytochemistry we show that in DRG cultured neurons PKCε translocation induced by endothelin-1 was prominently seen in a peptidergic subpopulation of cultured DRG neurons largely negative for isolectin B4 staining, indicating that in basal conditions functional expression of endothelin receptors does not occur in non-peptidergic, RET-expressing nociceptors. Translocation was blocked by the specific ETA-R antagonist BQ-123 while it was unaffected by the ETB-R antagonist BQ-788. No calcium response in response to endothelin-1 was observed in sensory neurons, while large and long-lasting responses were observed in the majority of non-neuronal cells present in DRG cultures, which are ensheathing Schwann cells and satellite cells, identified with the glial marker S-100. Calcium responses in non-neuronal cells were abolished by BQ-788. The fraction of peptidergic PKCε-translocated neurons was significantly increased by nerve growth factor, while in the presence of neurturin or glia-derived neurotropic factor (GDNF), an IB4-positive subpopulation of small- and medium-sized neurons showed PKCε translocation induced by endothelin-1 which could be blocked by BQ-123 but not by BQ-788. Our in vitro results show that the level of expression of functional endothelin receptors coupled to PKCε is different in peptidergic and non-peptidergic nociceptors and is modulated with different mechanisms in distinct neuronal subpopulations.

2011 - Nimesulide inhibits protein kinase C epsilon and substance P in sensory neurons - comparison with paracetamol. [Articolo su rivista]
Vellani, Vittorio; Franchi, S; Prandini, Massimiliano; Moretti, S; Pavesi, Giorgia; Giacomoni, C; Sacerdote, P.
abstract

In this paper we describe new actions of nimesulide and paracetamol in cultured peripheral neurons isolated from rat dorsal root ganglia (DRG). Both drugs were able to decrease in a dose-dependent fashion the number of cultured DRG neurons showing translocation of protein kinase C epsilon (PKCɛ) caused by exposure to 1 μM bradykinin or 100 nM thrombin. In addition, the level of substance P (SP) released by DRG neurons and the level of preprotachykinin mRNA expression were measured in basal conditions and after 70 minutes or 36 hours of stimulation with nerve growth factor (NGF) or with an inflammatory soup containing bradykinin, thrombin, endothelin-1, and KCl. Nimesulide (10 μM) significantly decreased the mRNA levels of the SP precursor preprotachykinin in basal and in stimulated conditions, and decreased the amount of SP released in the medium during stimulation of neurons with NGF or with the inflammatory soup. The effects of paracetamol (10 μM) on such response was lower. Nimesulide completely inhibited the release of prostaglandin E2 (PGE2) from DRG neurons, either basal or induced by NGF and by inflammatory soup, while paracetamol decreased PGE2 release only partially. Our data demonstrate, for the first time, a direct effect of two drugs largely used as analgesics on DRG neurons. The present results suggest that PKCɛ might be a target for the effect of nimesulide and paracetamol, while inhibition of SP synthesis and release is clearly more relevant for nimesulide than for paracetamol mechanism of action.

2001 - Enantiomeric separation of local anaesthetic drug by HPLC on chiral stationary phases [Abstract in Atti di Convegno]
Rustichelli, Cecilia; Ferioli, Valeria; Pavesi, Giorgia; Gamberini, Gianfranco
abstract

The present communication deals with the stereoselective separations of the enantiomers of articaine hydrochloride and its metabolite articainic acid by HPLC on chiral stationary phases.