Relazione annuale

Background: University students are considered a population class with a high risk of nutritional deficiencies due to the bad eating habits which assume when they begin their university studies [1], with the consequent increase in this population class of the risk of malnutrition and/or obesity, metabolic and cardiovascular diseases [2]. Over the past 20 years there has been an increase in obesity rates among university students, therefore they should be seen as a group that requires special attention regarding health promotion, especially food [3]. The interest in a healthy diet can lead to a psychological obsession known as orthorexia [4], frequent among students in the biomedical field and in the sports context [5,6]. The high levels of stress recorded in university students have been related to the use of drugs to enhance their cognitive abilities [7]. The phenomenon of self-medication is one of the main problems for public health, with high levels recorded among students of health professions [8]. A high adherence to the Mediterranean diet seems to bring significant and different health benefits [9], with an impact even on the composition of the intestinal microbiota [10]. Although several studies have investigated the eating habits of Spanish university students [11,12], little is known about the daily energy and nutrient intake among Italian university students. Furthermore, it would be interesting to evaluate the use of drugs and nutraceuticals, including the tendency to self-medication between Italian and Spanish students of biomedical faculties, where higher rates of orthorexia are registered. In addition to this, although several studies have investigated the effect of the Mediterranean diet on the intestinal microbiota [10,13,14], little attention has been paid to the effect of this diet on the oral microbiota, one of the most relevant microbial habitats from a clinical point of view. It is therefore interesting to analyse and investigate these aspects.
Objective: The aim of this study is to evaluate self-medication, adherence to the Mediterranean diet, the relationship between lifestyle and biomarkers of the metabolic and immunological status, and impact of eating habits on the oral microbiota composition.
Methods: Students, doctoral students, post-docs and specialists in the biomedical and pharmaceutical fields will be recruited in Italy (N = 200) and in Spain (N = 200). Data will be collected through questionnaires in order to evaluate self-medication, eating habits, level of physical activity, orthorexia and lifestyle of the subjects. The entire group will be evaluated with clinical parameters of metabolic status and the quality of saliva and urine. The latter will be determining factors for the selection of 50 Italian and 50 Spanish students, on which parameters of immunological and antioxidant status, cortisol, urinary phenols, and the composition of the oral microbiota will be evaluated.
Results: Italian and Spanish students may have different eating habits and lifestyles. It is assumed that subjects with high adherence to the Mediterranean diet have a better metabolic and immunological status. Moreover, high rates of orthorexia are expected among students who practice sports. The composition of the oral microbiota could vary depending on the type of diet and consistently with the immunological status markers.

Introduction: The International Association for the Study of Pain (IASP) define pain as “An unpleasant sensory and emotional experience associated with, or resembling that associated with, actual or potential tissue damage” [1]. When pain is experienced it can be acute, persistent, or in extreme cases chronic [2]. Irrespective of the type of pain whether it is acute or chronic pain, the underlying origin is inflammation and the inflammatory response [3]. In particular, chronic pain due to tissue inflammation, nerve lesion, tumor invasion, or chemotherapy represents a major health problem in the health care system [4]. Chronic pain is among the most common complaints in outpatient clinic [5]. It is estimated that 11-40% of adult population suffers from chronic pain [6]. Moreover, chronic pain is usually accompanied with emotional changes, including anxiety, depression, or even suicidal tendencies [7].
Pain’s highly individual and subjective nature is one of the factors that make it so difficult to deline objectively and to treat clinically [8]. Despite the availability of several analgesic and anti-inflammatory options, the effective treatment of pain and inflammation is still a challenge for clinicians, and the balance between efficacy and safety aspects remains both crucial and difficult [9]. Thus, the identification of new potential targets which may affect pain and inflammatory processes is becoming an urgent clinical and therapeutic need.
Aim: This scientific project derives from a close collaboration with various national and foreign research groups, in order to establish an efficient and highly multidisciplinary approach to investigate the pharmacological profile of new compounds and herbal derivatives with significant antinociceptive and antinflammatory effects to be used for the treatment of pain and inflammation. Particularly, this thesis reported the in vivo studies carried out at National Center for Drug Research and Evaluation of Istituto Superiore di Sanità in Rome.
Materials and Methods: The research approach involves the in vivo studies of natural (ammonium glycyrrhizinate; rubiscolin-6 and soymorphin-6) and non-natural compounds (C-terminal amides derivatives of rubiscolin-6 and soymorphin-6; six analogues of endomorphin-2 and DAPEA; two compounds obtained in silico studies; a series of lonidamine with high CB1 receptor affinity and new pH-sensitive formulation of niosomes containing Polysorbate 20 derivatized by Glycine and loaded with ibuprofen) using murine models of pain and inflammation. All experiments were carried out using male CD-1 mice weighing 25 g. The Service for Biotechnology and Animal Welfare of the Istituto Superiore di Sanità and the Italian Ministry of Health authorized the experimental protocol according to Legislative Decree 26/14.
Results: The results suggest the potential use of ammonium glycyrrhizinate (AG) for clinical treatment of pain and/or inflammatory-related diseases, included diabetic peripheral neuropathy. In fact, AG exerts long- lasting antinociceptive effects, since AG-induced antinociception was observed until 24–48h after a single administration [10]. Furthermore, a short-repeated treatment with AG was able to contrast significantly neuropathic hyperalgesia induced by streptozotocin (STZ) injection [11].
The results obtained by in vivo studies testing two natural compounds, rubiscolin-6 and soymorphin-6, and their C-terminal amides derivatives, show that rubiscolin-6 was able to increase the nociceptive threshold to thermal stimuli after supraspinal administration, but was ineffective after subcutaneous administration, in an experimental paradigm of chemical-induced nociception as the formalin test. On the other hand, instead, its derivatives (rubiscolin-6 C-amide) centrally administered demonstrates a strong antinociceptive effect higher than rubiscolin-6, and it is effective after subcutaneous administration. Both soymorphin-6 and its derivative soymorphin-6 C-amide induced a robust antinociceptive effect after central administration in tail flick test but both peptides have not been able to change the behavioral response to chemical-induced nociception in the formalin test [12].
The in vivo antinociceptive profile of new six tetrapeptide models containing α-amino-γ-lactam of Freidinger in position 2 and 3, as analogues of endomorphin-2 (EM-2) and DAPEA, revealed that one of them (peptide A2D) exhibited a strong antinociceptive effect in vivo after intracerebroventricular administration, performing better than the parent compounds EM-2 and DAPEA. Peptide A3D is also able to produce antinociceptive effect both in the acute and in the inflammatory phase of the formalin test [13]. The in vivo results obtained from two compounds (tripeptides 6 and 11) derived from previous in silico studies conducted on a virtual screening workflow of a library consisting of ~6 million molecules with the aim to find potential lead compounds that could manifest activity on the KOR, revealed their ability to induce an antinociceptive effect after intracerebroventricular and subcutaneous administrations in the tail flick and formalin tests, respectively [14].
Subsequently, it was analyzed the in vivo orexant/anorexant and antinociceptive profile of a series of lonidamine joined Leu-, tert/Leu- and Val-amino acids with different C-terminal functional groups (LONI 1-4,11) as novel compounds with high CB1 receptor affinity and selectivity with different biological activity depending on the C-terminal substitution and amino acid residues and endowed with activities. The results demonstrated that LONI2 and LONI4 were able to inhibit food intake (anorexant effect) consistent with an inverse agonism at CB1 receptors. On the other hand, LONI11, an agonist towards cannabinoid receptors CB1 and CB2, proved to carry out a significant orexant an antinociceptive effect at the central and periphery levels [15].
Finally, a new pH-sensitive formulation of niosomes containing Polysorbate 20 derivatized by Glycine and loaded with ibuprofen (NioIbu) was in vivo tested, using models of pain and inflammation. The results demonstrated that NioIbu, administered 2h before testing, reduced nociception, whereas the free form of ibuprofen was ineffective. In a model of inflammatory pain (hyperalgesia induced by zymosan) NioIbu induced a long-lasting reduction in hyperalgesia in treated mice. In a model of neuropathic pain induced by sciatic nerve chronic constriction, NioIbu reduced both neuropathy-induced allodynia and hyperalgesia [16].
Conclusions: Taken together, all these findings indicate that this experimental work allowed us to find interesting novel compounds and to deepen the potential of herbal derivatives. In conclusion, these findings create prospects for conducting clinical studies with the compounds presented, which show in fact their effectiveness for chronic pain and inflammation control

Edoardo Pisa conducted a series of experiments aimed at demonstrating that specific nutrients of maternal milk are pivotal in the developmental regulation of cognitive capabilities and underlying neural substrates. The candidate Edoardo Pisa conducted a series of elegant studies in which he demonstrated that mice reared in the absence of two specific oligosaccharides, during lactation, results in consistent impairments in attention, memory and executive functions. Additionally, his studies identified some of the underlying molecular, genetic and microbiological determinants of the aberrant phenotype. During his PhD, he had the opportunity to present his results in several international conferences, also receiving several awards. Finally, Edoardo Pisa conducted a series of experiments, in which he investigated that exogenous compensatory supplementation of these oligosaccharides mitigated the onset of the aforementioned impairments. The exceptional value of his studies resulted in a top-ranking journal publication (Molecular Psychiatry), in which he has a lead role (Hauser J*, Pisa E*, et al. Sialylated human milk oligosaccharides program cognitive development through a non-genomic transmission mode. Mol Psychiatry. 2021 Jul;26(7):2854-2871. doi: 10.1038/s41380-021-01054-9. Epub 2021 Mar 4. PMID: 33664475. * indicates co-first authors), two manuscripts that have been recently submitted and two manuscripts currently in preparation. Throughout his PhD, he also spent four months abroad, at Goethe-Universität of Frankfurt am Main, as a guest PhD student, wherein his outstanding attitude and expertise have been highly appreciated by the lab head (Prof. Dr. David Slattery).

Far from being limited to weight gain, obesity is now associated with a cluster of central and peripheral alterations including modification in the integrity and functionality of the blood-brain-barrier (BBB), systemic low-grade inflammation and fatty liver diseases. In the last two decades, oleoylethanolamide (OEA), a naturally occurring bioactive lipid belonging to the family of N-acylethanolamides (NAEs), has received great attention for its biological properties which spanning in different domains: from neuroprotection to memory, from inflammation to mood disorders, from the regulation of satiety to glucose homeostasis and lipid metabolism. Over the 3 years of experimental activity as a PhD student, Marzia Friuli has investigated, under my supervision, the effects of the peripheral administration of oleoylethanolamide (OEA) on the alterations related to obesity and eating disorders by focusing her attention on: 1) the analyses of the expression of protein involved in the integrity and functionality of the BBB, 2) the possible role of OEA in adipogenesis and on parameters of oxidative and endoplasmic reticulum stress.
To this aim, diet-induced obesity (DIO) rats were obtained by exposing male rats to a high-fat diet (HFD: 60% of the Kcal from fats) for 11 weeks; control rats received a low-fat diet (LFD: 10% of the Kcal from fats). Thereafter OEA was intraperitoneally (i.p.) administered daily, for 2 weeks, at the dose of 10 mg/kg. Through different molecular biology techniques, Marzia has shown that OEA affects the expression of key protein involved in the integrity and functionality of the BBB, including vimentin and zona occludens-1. Moreover, in collaboration with Prof.ssa Anna Maria Giudetti of University of Salento, Marzia has shown that OEA reduce the adipogenesis and ameliorates parameters of oxidative and endoplasmic reticulum stress in the liver of HFD-fed rats. Therefore, the results obtained by Marzia suggest neuroprotective and hepatic antiadipogenic and antioxidative effects exerted by OEA and adds novel information on the anti-obesity properties of this compound.
It has also been demonstrated that the pro-satiety effect of OEA is associated to the activation of different central pathways, including the oxytocinergic and the histaminergic systems. The neuropeptide oxytocin, by binding its receptors (oxytocin receptor, OXY-R), regulates a variety of physiologic functions, including eating and metabolism. Several studies have demonstrated an interplay between the oxytocinergic system and the endogenous NAEs (such as anandamide, oleoylethanolamide and palmitoylethanolamide) in the modulation of both homeostatic and non-homeostatic aspects regulating food intake.
In this context, Marzia evaluated whether rats with different history of aberrant food intake showed altered OXY-R expression in specific brain areas and then whether an increase of acylethanolamides tone might affect this altered OXY-R expression. To this aim 2 different animal models of aberrant eating were used:
1. a rat model of binge-like eating was used in in collaboration with Prof. Carlo Cifani of University of Camerino. In such model after cycles of intermittent food restrictions/refeeding and palatable food consumptions, female rats show a binge-like intake of palatable food, following a 15-min exposure to their sight and smell ("frustration stress") ;
2. a rat model of diet-induced obesity based on hedonic overfeeding, induced by the exposure to a cafeteria-diet (CAF rats) for 40 days and subsequently to an abstinence period from this diet.
The impact of the stimulation of the endogenous acylethanolamide tone on the OXY-R expression was evaluated pharmacologically, by peripherally administering OEA (10 mg/kg i.p.) to bingeing rats and by administering the fatty acid amide hydrolase (FAAH, the enzyme involved in the catabolism of the acylethanolamides) inhibitor, PF-3845 (10 mg/kg subcutaneously), to CAF rats.
The results obtained by Marzia revealed that aberrant eating behavior was associated with a reduced OXY-R expression and that this hypo-functionality of the oxytocinergic system was restored by OEA and PF-3845 treatments. Therefore, the modulation of the central oxytocinergic signaling by increasing acylethanolamines tone might represent one of the mechanisms that coordinate energy balance at the crossroads between homeostatic and non-homeostatic mechanisms in the treatment of aberrant eating behaviors.
Finally, an interplay between OEA and the oxytocinergic and histaminergic systems has been extensively demonstrated. Indeed, OEA, to induce satiety, stimulates oxytocin neurosecretion from the paraventricular nucleus (PVN) and this occurs in a histamine-dependent way. In collaboration with Prof.ssa Beatrice Passani of University of Firenze, Marzia explored the relationship between histaminergic system and OEA on the modulation of oxytocin immunoreactivity in the PVN of chronic social defeat stress (CSDS) animal model. Both histidine decarboxylase null (HDC- /-) and HDC+/+ mice were subjected to CSDS for 21 days and treated with either OEA or vehicle daily, starting 10 days after CSDS initiation, until sacrifice. Undisturbed mice served as controls. The results obtained by Marzia revealed that, in this animal model, the beneficial effects of OEA occur only in the presence of a functioning histaminergic system.

During his 3-years PhD attendance period, the candidate Alessandro Zappalà both worked on a project from our own department and managed to extend the scope of our collaboration with Kobe University’s department of Pharmacology.
During the first half of his period, he tried to understand if prokineticin 2 (PK2) may have an involvement in the antinociceptive effect of vortioxetine. PK2 is a new chemokine that, in different animal models of neurophatic pain, is strongly up- regulated and induces hyperlgesia
Vortioxetine is a well-known antidepressant that, working on serotoninergic pathways, has been shown to fight neuropathic pain.
Alessandro Zappalà participated to elucidate the molecular mechanisms behind the antinociceptive effects of vortioxetine demonstrating, by Western Blot analysis, that mice with Chronic Constraint Injury (CCI) and treated with vortioxetine expressed lower levels of the dimeric form of prokineticin 2 (PK2) in their medial prefrontal cortex (mPFC), explaining, at least in part, the antinociceptive effect of vortioxetine.
In his collaboration with Kobe University, he examined the changes in activity in mPFC in a murine model of depression: mice were injected intracranially with a viral vector expressing the fluorescent calcium sensor GCaMP7c and implanted with an optic fibre in order to measure the fluorescence emitted by the sensor in response to increased neuronal calcium release; after recovery from surgery, half of the mice underwent chronic social defeat stress (SDS) from an aggressive mouse while the other was left untouched; finally all the mice were tested for depressive-like behaviour using female urine sniffing test (FUST) and their mPFC activity was recorded via optic fibre. During FUST, neuronal activity of unstressed mice remained constant whether they were sniffing water or urine, while in stressed mice such activity becomes more variable during urine sniffing, even if stressed mice display much fewer sniffing episodes than control mice.

Durante il secondo anno e buona parte del terzo anno il Dott. Marchetta si è occupato di valutare come gli eventi stressanti subiti durante l’adolescenza potessero influenzare gli aspetti cognitivi ed emozionali e la suscettibilità ad altri eventi stressanti in età adulta. E’ noto, infatti, che l'adolescenza è un periodo in cui la struttura del cervello è in continua maturazione ed evoluzione. Pertanto, eventi stressanti che si presentano durante questa finestra di sviluppo potrebbero avere effetti più dannosi rispetto a quelli sperimentati in età adulta. Abbiamo, così, valutato se l'esposizione al social defeat stress (SDS), un modello animale di stress sociale nei roditori, durante l'adolescenza e/o un singolo stress sperimentato in età adulta, influenzi lo sviluppo di alterazioni emozionali e cognitive e se le alterazioni comportamentali siano legate a qualsiasi modifica dell'espressione del BDNF nell'ippocampo e dei livelli plasmatici di corticosterone. Abbiamo dimostrato che l'esposizione all’SDS durante la prima adolescenza influenza la capacità di far fronte a una seconda sfida vissuta più avanti nella vita. In particolare, i ratti esposti a SDS durante la prima adolescenza e poi a SPS in età adulta hanno mostrato resilienza contro lo sviluppo di alterazioni emozionali, dell'arousal e della memoria spaziale, ma hanno dimostrato vulnerabilità nel richiamo della memoria avversiva. Inoltre, i ratti esposti a entrambi i fattori di stress hanno mostrato resilienza verso le alterazioni indotte dall'esposizione all’SDS sull'espressione del BDNF ippocampale e sui livelli plasmatici di corticosterone. Il solo SPS ha indotto alterazioni dell'attività locomotoria e della memoria spaziale a lungo termine che non sono state rilevate nei ratti che hanno sperimentato l’SDS durante l'adolescenza. Per quanto è a noi noto, questo studio è il primo a valutare se l'esposizione a un protocollo di stress precoce che mima uno stress sociale simile al bullismo negli adolescenti influisce sullo sviluppo di psicopatologie in età adulta in risposta a traumi aggiuntivi sperimentati più tardi nella vita. Questi risultati aprono la strada a futuri studi volti a indagare il ruolo di specifiche aree cerebrali e della neurotrasmissione che media i meccanismi di resilienza o vulnerabilità agli effetti dell’esposizione allo stress nei primi anni di vita.
Attualmente, uno degli stress che gli adolescenti sperimentano comunemente durante l’adolescenza è l’isolamento dalle diverse attività sociali. Ad esempio, studi clinici hanno dimostrato che quando gli adolescenti trascorrono molto tempo giocando ai videogiochi, riducono il tempo per interagire con amici o genitori, partecipando a sport e altre attività importanti per lo sviluppo delle loro abilità sociali. Inoltre, le misure di distanziamento fisico attualmente adottate per contenere la diffusione del COVID-19 hanno costretto molti adolescenti nel mondo a evitare qualsiasi forma di contatto sociale, e ciò potrebbe minare alla loro futura salute mentale. Pertanto, durante lo stesso periodo, il Dott. Marchetta ha valutato nel ratto se brevi periodi ripetuti di stress da isolamento sociale possono alterare l'emotività e la funzione cognitiva nei ratti adulti e se questi effetti sono dipendenti dal sesso. In aggiunta, il Dott. Marchetta ha esaminato se lo stress da isolamento sociale breve e ripetuto durante l'adolescenza e/o il singolo stress (SPS) in età adulta influenzino il successivo sviluppo di alterazioni emozionali e cognitive. Lo stress da isolamento sociale (SIS) è una procedura ben convalidata per riprodurre nei roditori alterazioni simili a quelle osservate in diversi disturbi psichiatrici. Il SIS viene generalmente eseguito nei ratti maschi a partire dall'adolescenza fino all'età adulta per periodi lunghi e continuativi. Tuttavia, gli effetti a lungo termine di un periodo più breve di esposizione al SIS durante la prima adolescenza e le potenziali differenze di sesso in risposta alla SIS non sono ancora stati studiati. I nostri dati hanno indicato che lo stress da isolamento sociale durante l'adolescenza causa ipereccitazione e un fenotipo ansioso solo nei ratti maschi e un’esacerbazione del comportamento di evitamento nelle femmine in età adulta. L’SPS ha indotto una riduzione dell'attività locomotoria in entrambi i sessi, disfunzioni della memoria spaziale nei maschi e alterate dinamiche della memoria avversiva nelle femmine. I ratti che hanno sperimentato entrambi i fattori di stress hanno mostrato resilienza verso le alterazioni emozionali indotte dall'esposizione allo stress da isolamento sociale nei maschi e verso il comportamento di evitamento indotto dalla SIS nelle femmine. L’SPS ha alterato l’attività locomotoria e la ritenzione della memoria spaziale; questi effetti sono risultati assenti nei ratti esposti a SIS e successivamente esposti a SPS. Pertanto, è importante evidenziare che brevi e ripetuti periodi di isolamento sociale durante la prima adolescenza potrebbero causare importanti effetti a lungo termine in modo differente in base al genere e che l’isolamento sociale breve e ripetuto potrebbe alterare la resilienza verso alterazioni della sfera emozionale e cognitiva quando un evento traumatico si verifica più tardi nella vita. Il relativo manoscritto è attualmente in fase di stesura.
Durante il terzo anno il Dott. Marchetta si è occupato di scrivere una review su invito dal titolo: “Combining exposure- based therapies with pharmacology: from preclinical stage to clinical interventions in PTSD treatment”, attualmente in fase di sottomissione. Il Dott. Marchetta ha, altresì, raccolto dati preclinici da diverse banche dati, riguardanti l’effetto dello stress sulla fear extinction al fine di valutarne gli effetti complessivi in una metanalisi attualmente in preparazione .
Oltre l’attività di ricerca il Dott. Marchetta ha partecipato, durante al secondo anno, al progetto “Smart drugs? No grazie” organizzato dall’ASL Roma 6, come docente presso istituti di scuole secondarie di primo e secondo grado.

Poly (ADP-ribose) polymerase (PARPi) inhibitors are nicotinamide analogues that compete with NAD+ for the binding to the catalytic domain of PARP1, an enzyme able to bind DNA, acting as a sensor of DNA damage, and to activate DNA repair mechanisms. As monotherapy, PARPi exert selective cytotoxic effects in tumors with altered DNA repair, according to a model of synthetic lethality; in combination with standard chemotherapy, they increase the sensitivity of cancer cells to DNA damaging agents. PARPi have been previously approved as anticancer treatment of BRCA1/2 (components of the homologous recombination system, HR) mutant, advanced or metastatic solid tumors (ovarian, breast, prostate and pancreatic cancer). Recently, their use has also been extended to tumors with HR-deficiency not directly related to mutations in the BRCA genes. Data obtained in our laboratory have demonstrated the efficacy of the PARPi olaparib as a cytotoxic agent in cell lines and primary cultures of Acute Myeloid Leukemia (AML) and Myelodysplastic Syndromes (MDS). Given their oral bioavailability and tolerability profile and considering the genetic instability that characterizes hematological malignancies, PARPi might represent a potential therapeutic strategy for relapsing AML patients who cannot tolerate the adverse effects of standard chemotherapy. Among the AML cell lines tested, the NB4 cell line resulted the most sensitive to the treatment with olaparib. NB4 is a cellular model of human Acute Promyelocytic Leukemia (APL), a subtype of AML which accounts for 10-15% of all AMLs. The standard treatment of APL is based on the use of all-trans retinoic acid (ATRA) combined with anthracyclines or arsenic trioxide (ATO). However, 5-10% of patients develop relapsed/refractory disease, which has few therapeutic options.
Aims of the PhD project:
To clarify the role of PARPi olaparib in MDS: besides its involvement in DNA repair, the founding member of the PARP family, PARP1, has a crucial role in the regulation of gene transcription through post-translational modification of proteins (PARylation). The involvement of PARP1 in the modulation of gene expression suggests additional therapeutic implications of PARPi in MDS. On this basis, Dr. Giansanti contributed to demostrate that olaparib exerts differentiating effects in primary cultures of human bone marrow samples collected from MDS patients. Cell differentiation was evaluated by morphological (May Grunwald-Giemsa staining) and immunophenotypic (flow cytometer) analysis and by quantitative analysis (qRT-PCR) of transcription factors relevant to myeloid cell differentiation (PU.1 and CEBPA). The results indicated that olaparib induced a decrease of CD117+ or CD34+ hematopoietic progenitors and increase of mature CD11b+/CD16+ and CD10+/CD15+ neutrophils in almost all MDS samples together with an increase of PU.1 and CEBPA. Moreover, olaparib was tested in combination with the hypomethylating agent decitabine, which is FDA-approved for all MDS subtypes. Noteworthy, the combination of olaparib and decitabine significantly decreased the percentage of immature myeloid progenitors and increased that of differentiated neutrophils. This effect was paralleled by the up-regulation of PU.1 and CEBPA transcription factors. These findings suggest that olaparib, as single agent or in combination with decitabine, might have a therapeutic potential in MDS especially for patients ineligible for chemotherapy or bone marrow transplantation.
To identify new potential therapeutic strategies for ATO-resistant APL:
1) Generation of ATO-resistant APL clones: four NB4 clones were generated by limiting dilution (CL1, CL2, CL3, CL4) and then exposed to increasing concentrations of ATO in order to obtain the corresponding ATO-resistant clones (CL1-R, CL2-R, CL3-R, CL4-R). All ATO-resistant clones maintained the sensitivity to ATRA. To maintain the resistant phenotype, the ATO-resistant clones were kept in culture with 1 µM of ATO, which is a clinically relevant concentration.
2) Study of the mechanisms involved in the resistance to ATO of APL clones:
- Since one of the mechanisms of ATO-resistance is the loss of the PML/RARA chimeric gene (pathognomonic of APL), the presence of the PML/RARA translocation was evaluated and confirmed by DNA analysis through PCR techniques and electrophoresis on agarose gel.
- Activation of extrinsic and intrinsic apoptosis by ATO has been reported in sensitive, parental NB4 cells. To study the mechanism of ATO resistance in APL clones, sensitive and resistant cells were treated with ATO and analyzed by western blotting for proteins involved in apoptosis. ATO induced a dose-dependent PARP1 cleavage and activation of caspase 3 and 8 only in ATO-sensitive clones. Moreover, ATO induced a high level of histone 2AX (γH2AX) phosphorylation at serine 139 (a DNA double stand break marker) only in ATO-sensitive clones.
- Following these preliminary data, a study of RNA expression of genes involved in the apoptotic pathway of APL clones was performed by qRT-PCR. APAF1, NOL3, BCL2 and BIRC3 genes resulted up-regulated in the ATO-resistant clones, while the CD70 and IL10 genes were down-regulated. Furthermore, a direct correlation was observed between the high BCL2 gene expression and the response to the BCL2 inhibitor venetoclax treatment, suggesting the efficacy of venetoclax in the case of APL with high BCL2 gene expression. However, the gene that was found strongly up-regulated in ATO-resistant clones was BIRC3 (cellular IAP2). Future studies are required to elucidate the efficacy of the “Smac mimetics” family of drugs that target the cellular IAPs for overcoming APL resistance to ATO.
3) Analysis of the cytotoxic effects of PARPi on ATO-resistant APL: experiments were conducted to evaluate the efficacy of four clinically approved PARPi olaparib (1.25-10 µM), niraparib (1.25-10 µM), talazoparib (12.5-100 nM), rucaparib (1.25-10 µM) and of the clinically investigated veliparib (5-20 µM) in the ATO-sensitive and -resistant clones. Cytotoxicity was evaluated by the MTS metabolic assay. For each drug, the inhibitory concentration 50 (IC50) was calculated. All ATO-resistant and-sensitive clones were sensitive to olaparib, niraparib and talazoparib with IC50 values below or within the range of plasma peak concentrations reached in cancer patients. Conversely, rucaparib and veliparib showed IC50 values higher than the plasma peak concentrations. Based on these results, experiments aimed at testing drug combinations with PARPi were performed using olaparib, niraparib or talazoparib.
4) Analysis of the cytotoxic effects of PARPi, in combination with demethylating agents, against ATO-resistant APL:
- The PARPi olaparib, niraparib and talazoparib were initially tested in combination with agents endowed the antileukemic activity and whose mechanism of action involves a DNA damage response with PARP1 intervention. In particular, PARPi were associated with the hypomethylating agents azacitidine and decitabine (0.125-1 µM) and with high-dose vitamin C (ascorbate, 0.125-2 mM). Cytotoxicity was evaluated by the MTS viability test or by trypan blue dye exclusion count. For each drug IC50s were calculated. All the APL clones were highly sensitive to azacitidine and decitabine and to high-dose vitamin C. Then, drug combination experiments were performed, and synergism was evaluated by the CompuSyn Software.
- PARPi plus hypomethylating agents: decitabine and azacitidine are inhibitors of DNA methyltransferase (DNMT), both used in clinical practice for AML patient ineligible for intensive chemotherapy and MDS. Both drugs are cytidine analogs that cause DNA damage as a consequence of their random incorporation into DNA (azacitidine also in RNA). The PARPi olaparib, niraparib or talazoparib in combination with azacitidine or decitabine induced strong synergistic cytotoxic effects in ATO-resistant APL cells, without differences related to the PARPi used. These effects were likely the result of increased DNA damage and it is reasonable to hypothesize that similar PARPi and hypomethylating agent interactions, reported in others tumors, may also occur in APL cells: altered processing by BER and PARP1 of the aberrantly incorporated cytidine analogue and trapped DNMT1; induction of a BRACness phenotype by down-regulating the expression of DNA repair enzymes; accumulation of ROS with consequent DNA damage that triggers PARP1 activation and becomes deleterious in the presence of PARPi; increased drug retention at the DNA damage sites.
- PARPi plus high dose vitamin C: high dose vitamin C, in the form of ascorbate, has been shown to promote 5-hydroxymethylcytosine (5hmC)-mediated DNA demethylation by enhancing the activity of Ten-Eleven Translocation (TET) enzymes and triggering a DNA damage response. On this basis, the candidate helped to hypothesize that PARPi may increase the sensitivity of APL cell lines to DNA damage induced by ascorbate. The synergistic effect was observed at concentrations of ascorbate that did not induce the production of reactive oxygen species but was associated with a significant increase in 5hmC (marker of DNA demethylation) and γH2AX levels, as a result of ineffective BER-mediated processing of the oxidized intermediates associated with DNA demethylation. The ascorbate combination with olaparib exerted synergistic effects only in three out of the eight clones, while its combination with niraparib resulted in additive effects in two clones and synergistic effects in all the other clones. Noteworthy, the ascorbate/talazoparib combination was highly effective in all ATO-sensitive and -resistant clones. These results indicate talazoparib as the best candidate for combined with ascorbate.
- Mechanism of action of talazoparib plus high dose vitamin C: PARPi exert their activity by inhibiting the catalytic domain of PARP1 and through the binding of PARP1 (trapping) in DNA. Among the different PARPi, talazoparib has the highest PARP1 trapping activity in DNA compared to the other PARPi. In ATO-resistant APL cells, the talazorarib/ascorbate combined treatment increased PARP1 levels in the chromatin bound protein fraction, indicating an increase of PARP1 trapping. Ascorbate increased 5hmC levels in DNA, which recruit PARP1 for 5hmC conversion into cytosine. On the other hand, the combination with talazoparib enhances PARP1 entrapment on DNA creating unrepaired DNA lesions. The same effect was also demonstrated in a human melanoma cell line resistant to treatment with the BRAF inhibitor dabrafenib.

Le attività di ricerca sono state svolte presso il Centro di Riferimento per la Medicina di Genere dell’Istituto Superiore di Sanità. Tali attività sono parte integrante di un progetto di ricerca riguardante l’associazione dell’esposizione a residui di pesticidi e impatto sullo sviluppo puberale di bambine affette da telarca prematuro idiopatico, ossia sviluppo prematuro della ghiandola mammaria, in un’area prettamente agricola in cui è stata osservata un’incidenza della patologia.
Nello specifico le attività svolte per la Tesi sono state incentrate su: a) la caratterizzazione degli effetti di alcuni pesticidi maggiormente utilizzati in agricoltura, quali clorpirifos, imidacloprid e glifosto; b) la messa a punto di uno strumento per la valutazione dell’esposizione delle bambine attraverso la dieta. Entrambi questi aspetti convergono nelle fasi della valutazione del rischio tossicologico.
La valutazione degli effetti dei pesticidi è stata effettuata mediante studi in vitro, utilizzando due linee cellulari umane di ghiandola mammaria, MCF-7 di origine tumorale molto utilizzate in letteratura soprattutto per gli studi sul cancro, e MCF-12A meno utilizzate ma di origine non tumorale, pertanto, più adeguate allo scopo dello studio. La finalità ultima è stata quella di mettere a punto un modello più riproducibile la fisiologia in vivo della ghiandola mammaria. Infatti, a tale scopo è stato messo a punto un modello di studio tridimensionale (3D) utilizzando la linea cellulare di ghiandola mammaria umana non tumorale (MCF12A). Le due linee cellulari sono state trattate con concentrazioni derivate da valori reali di esposizione nei bambini come riportato dai dati epidemiologici presenti in letteratura. Gli effetti dei pesticidi sono stati valutati attraverso una batteria di tests attraverso i quali comparare la risposta delle due linee cellulari. Tali tests comprendono la valutazione della citotossicità, l’induzione di apoptosi/necrosi, la produzione di ATP, l’analisi dei ROS, la produzione di estradiolo e l’analisi dell’espressione genica di specifici recettori nucleari coinvolti nello sviluppo della ghiandola mammaria.
I risultati dello studio dimostrano che i tre pesticidi testati possono influenzare l’attività metabolica cellulare e l’espressione genica dei recettori nucleari in entrambe le linee cellulari ma con capacità ed entità di risposta diverse. Gli effetti osservati possono essere messi in relazione ad alterazioni critiche dei processi cellulari coinvolti nello sviluppo della ghiandola mammaria che possono portare ad un evento patologico in fase precoce della vita del bambino.
Nell’ambito dello studio epidemiologico, le attività svolte hanno condotto alla messa a punto di un questionario di frequenza alimentare diviso in gruppi di alimenti che riportasse per ognuno di essi la quantità consumata, la frequenza di consumo e il luogo di acquisto. In questo modo il questionario rappresenta uno strumento con una doppia funzione, sia quella di rendere possibile il campionamento di alimenti prodotti localmente che quella di valutare l’esposizione attraverso la dieta. In questa fase di studio, l’elaborazione delle risposte ottenute ha reso possibile l’attuazione di un piano di campionamento degli alimenti prodotti a livello locale, in aziende o in orti privati divisi per stagionalità. Dai dati di analisi quantitativa di pesticidi eventualmente presenti si potrà valutare l’esposizione attraverso la dieta nelle bambine coinvolte nello studio.

Dr. Nico Antenucci in the three-year period of his PhD course developed and carried out research projects (see below) in the Lab of Molecular Neuropharmacology of IRCCS Neuromed in Pozzilli. His interest has been focused on molecular mechanisms underlying chronic pain, on the involvement of metabolites of the kynurenine pathway in pain and schizophrenia, on the molecular determinants of the pathological phenotype in mouse models of type-1 spinocerebellar ataxia, and on the role played by mGlu3 receptors in neuroinflammation. Dr. Antenucci has been highly productive, and he is co-Author of 5 publications in peer-reviewed International Journals, one manuscript under revision in the Journal of Neuroscience, and two posters presented in International Meetings. In addition, Dr. Antenucci is first co-Author in a manuscript in preparation describing the molecular basis of early cognitive dysfunction in SCA-1 mice.
Cinnabarinic acid (CA) has been and still is one of the main interest of Dr. Antenucci in the lab. He contributed to demonstrate that CA levels are reduced in the prefrontal cortex of patients affected by schizophrenia, and that systemic CA administration produces antipsychotic-like effects in mice. In addition new unpublished findings demonstrate that CA causes analgesia in models of inflammatory and neuropathic pain. These latter findings were obtained in collaboration with Prof. Sabatino Maione and Livio Luongo of the University of Naples. Dr, Antenucci has also applied optopharmacology to the study of the pain matrix, showing under the tutorship of Dr. Serena Notartomaso, that mGlu5 receptor blockade in the thalamus and prefrontal cortex is both sufficient and necessary for the analgesic activity of mGlu5 receptor negative allosteric modulators. Dr. Antenucci has also contributed to demonstrate that mGlu3 receptors support the maturation of parvalbumin-positive interneurons in the prefrontal cortex and drive microglial cells towards an anti-inflammatory phenotype. The latter demonstration was obtained in a mouse model of intrauterine growth restriction in collaboration with Prof. Oliver Baud (Sorbonne University, Paris). Finally, Dr. Antenucci has been involved in a study on the effect of transient mild hypoxia on biochemical and behavioral changes caused by chronic unpredictable stress in mice. The experimental paradigm mimicked the mild hypoxic conditions found in the air cabin during domestic flights.-

Nel progetto di ricerca, svolto come tesi di Dottorato, sono stati monitorati i dati di vendita di integratori alimentari contenenti botanicals (estratti e preparati vegetali), presso due farmacie territoriali di Roma. L’obiettivo è stato rivolto a prodotti di derivazione vegetale, con indicazioni d’uso specifiche: dislipidemie e controllo del peso corporeo. Negli ultimi anni, questi prodotti sono stati oggetto di un maggiore numero di sospette reazioni avverse sul territorio nazionale. Per avere una conoscenza reale della loro sicurezza d’impiego, si è voluto conoscere il “denominatore” relativo alle vendite e rapportarlo alle sospette reazioni avverse. Il progetto ha coinvolto figure professionali dalle responsabilità e competenze diverse, tra cui farmacisti del territorio laziale e Istituto Superiore di Sanità. Contestualmente sono state raccolte le segnalazioni di sospette reazioni avverse riguardanti preparati vegetali utilizzati nelle dislipidemie. Non è stato invece possibile per quelli utilizzati nel controllo del peso corporeo in quanto dati confidenziali, oggetto di ulteriori studi e relative pubblicazioni. Inoltre, un questionario sulla Fitovigilanza è stato indirizzato alle farmacie della Regione Lazio.
Questo è il primo lavoro di ricerca scientifica che rende disponibili dati di vendita di prodotti contenenti piante medicinali, provenienti da farmacie del territorio. La metà degli integratori venduti nelle due farmacie è costituito dai preparati vegetali. Ciascuna delle due categorie di interesse rappresenta il 9% degli integratori contenenti botanicals. Considerando l’uso crescente di questi prodotti, l’acquisizione altamente innovativa di questo tipo di dato, rappresenta un avanzamento delle conoscenze, rispetto allo stato dell'arte riguardante la sicurezza d’impiego.
Dai dati ottenuti, i seguenti elementi d’interesse spiccano in maniera evidente:
(i) elevata numerosità di formulazioni con la stessa indicazione d’uso
(ii) preponderante percentuale di composti multi-ingrediente
(iii) vasta gamma di piante contenute, sebbene non aventi funzioni fisiologiche in linea con quanto previsto dalle linee guida del Ministero della Salute
(iv) carenza di standardizzazione di alcuni estratti vegetali
(v) bassa partecipazione nel segnalare le sospette reazioni avverse
Complessivamente, i risultati di questo lavoro di Tesi sottolineano per entrambe le categorie di interesse, la limitata conformità tra i preparati vegetali in commercio e la ricerca scientifica su di essi. Ciò che viene immesso in commercio spesso non corrisponde a ciò che esiste nella letteratura scientifica. La mancanza di una composizione biochimica simile e costante non supporta la riproducibilità delle attività biologiche e farmacologiche. Quindi, la sicurezza non può essere garantita. Neanche la limitata conformità tra gli integratori multi-ingrediente commercializzati e il quadro normativo relativo, gioca a favore del profilo di sicurezza. I rischi attribuiti all'uso di preparati vegetali multi-ingrediente, non standardizzati, per le dislipidemie e per il controllo del peso corporeo dovrebbero essere evidenziati al fine di consentirne l'uso razionale e aumentarne i benefici.
Questo studio contribuisce anche a divulgare l'importanza del Sistema Italiano di Fitovigilanza, la quale risulta poco utilizzata dai professionisti della salute, quali farmacisti, maggiormente formati a livello accademico e direttamente coinvolti nel counselling.

In termini di salute pubblica, la tesi evidenzia la necessità di una opportuna caratterizzazione e definizione dei botanicals. Al fine di fornire ai consumatori indicazioni su ciò che acquistano e consumano, l’accuratezza e il controllo delle informazioni devono essere garantite. L’implementazione del network comunicativo nel settore della sicurezza di impiego dei preparati vegetali aiuterebbe a proteggere il consumatore da rischi inutili (e spesso prevedibili) e porterebbe ad un contenimento (se non ad una riduzione) dei costi sociali ascrivibili alle reazioni avverse in termini di ricoveri, consulenze, spese mediche, ecc. Poiché gli integratori non possono (e non devono) vantare proprietà terapeutiche, ma sono utilizzati per mantenere uno stato di salute fisiologico, almeno la loro sicurezza intrinseca dovrebbe essere garantita.

The student completed two projects on developmental exposure to neurotoxic metals by using in mice. She contributed to conceptualization, methodology and formal analysis of her PhD project and performed all the investigative work as follows; mouse colony maintenance, preparation and follow-up of chemical solutions, colony breeding, reproductive follow-up, behavioural experiments, dissections and sample collection/storage. The behavioural experiments that she applied in her PhD project included maternal behaviour assessment, neonatal behaviour battery (spontaneous movement test, homing test), open field test, three- chamber social interaction test, Morris water maze, olfactory habituation/dishabituation test, novel object recognition test, social recognition test, pre-pulse inhibition test. Preparation of experimental settings, organization, execution, video analysis and statistical analysis steps were completed by the student. During these 3-years, the student used software programs dedicated for behavioural analysis such as Any-Maze (Stoelting Co.) and Observer XT (Noldus). The PhD project results supported the literature on the effects of lead and showed detrimental effects of developmental, low-level lead exposure on short-term and long-term behaviours. Manganese – lead mixture study did not evidence any additive or synergistic effects of the two metals but highlighted specific effects of the single metals per se.

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