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Pharmacology, biochemistry and biomedical applications of plant stilbenes
Full text linkThis thesis reports the research I conducted on aspects of the pharmacology and biological activities of two natural stilbenes, Resveratrol (Rv) and Pterostilbene (Pt), major polyphenolic components of grapevines and blueberries respectively.
Over the years, these two molecules have drawn attention from the scientific community thanks to their beneficial bioactivities, relevant for many areas of health care. Numerous papers describe their protective roles against the metabolic syndrome, cancer development and neurodegeneration. These striking effects nowadays are not exclusively attributed to their redox properties but also to their capacity to modulate, directly or indirectly, the activities of key proteins of interconnected cellular signalling networks. Importantly, no noxious side effects have been reported. However, in spite of their positive features, Rv and Pt are not free of shortcomings. They are subjected to an intensive phase II metabolism, largely limiting their bioavailability. Moreover, while much research has been carried out to elucidate the molecular mechanisms of action of Rv, those of Pt still largely remain to be established. During my PhD program, I addressed both these themes. I describe below: 1) the attempts to increase the bioavailability of Rv and Pt 2) my investigations on intracellular processes affected by Pt as well as the demonstration of its therapeutic potential in two different in vivo models
1) A variety of approaches has been developed to overcome the poor bioavailability of many drugs. My research group turned to the development of prodrugs for oral administration. In the case of polyphenols, a useful prodrug implies the use of protecting groups to mask the characteristic hydroxyl groups and to avoid metabolic transformations by phase II conjugative enzymes. These substituents, technically defined as pro-moieties, are attached to the backbone of the molecule through a chemical bond which can be broken in the relevant physiological environment. The choice of the pro-moiety is important for the process of absorption from the intestinal tract. The type of linkage is crucial to achieve “cargo” regeneration with appropriate kinetics. We created prodrugs for oral administration. Thus, they ought to exhibit relative stability in the gastro-intestinal tract and lability in other biological compartments (body fluids and organs). When I joined the group of Dr. Mario Zoratti, N,N-disubstituted carbamoyl derivatives of Rv bearing a PEG 350 or a sugar pro-moiety had been created. Although they increased the poor water solubility of the natural compound, these prodrugs were too stable under physiological conditions. Thus, we moved to the N-monosubstituted carbamoyl bond, expected to be less stable, for new prodrugs. Importantly, this type of chemical bond is essentially stable in acid, while it can be more easily lysed at neutral or basic pH values. A first set of compounds incorporated amino acids as pro-moieties. We hypothesized that in addition to increasing water solubility, amino acids might be recognized and transported by specific carrier systems, expressed in the enterocytes, and thus favor the intestinal absorption of the molecules. In another set of Nmonosubstituted carbamoyl derivatives polyhydroxylated groups (dihydroxypropyl or 6-deoxygalactose) were attached to all three, two or one phenolic oxygen(s) of Rv. In this case, the hope was that sugar transporters might intervene to facilitate absorption through the intestinal epithelium. All synthetized compounds displayed hydrolysis kinetics suitable for their use as prodrugs. However, absorption after oral administration to rats turned out to be rather unsatisfactory, with no evidence of an involvement of membrane carriers. In no case did galactosylated Rv derivatives reach the bloodstream of rats. The best results were obtained by administering monosubstituted aminoacid derivatives. We speculate that the extended, three-pronged structure of the fully protecting prodrugs might interfere with recognition and transport. All synthesis procedures and the characterization and pharmacokinetic evaluation of these families of compounds have been already published. The papers are included in this thesis as chapters 1 and 2. Once completed the assessment of Rv prodrugs, I started a new research project involving Pterostilbene, a more compact and possibly more effective compound than Resveratrol. This represented the main theme of my PhD program investigations. As Pt was still poorly defined from a pharmacological point of view, we first evaluated its distribution in blood and organs after oral administration (see chapter 3). Thus, we developed an appropriate method for the quantification of the molecule and its metabolites in organs after administration to rats. The resulting protocol preserves stability and allows the quantitative extraction and clarification from biological matrices of Pt itself and its metabolites, and their quantitative analysis by HPLC/UV. We used this method to determine the levels of these compounds in the blood and several other organs as a function of time after administration as a single intra-gastric dose of 88µmoles/kg of body weight. Pterostilbene levels in several organs turned out to be much higher than those measured in blood, reaching nmoles/gr (μM)-range concentrations. Moreover, Pt-4’-Sulfate was identified as the major metabolic species. Its levels were higher than those of Pt in all tissues examined except the brain. In light of this, we next aimed to prevent metabolic modifications by phase II conjugative enzymes. I exploited the experience derived from the work with the prodrugs of Rv and in collaboration with the research group of Prof. Paradisi of the Department of Chemical Sciences of the University of Padua, we developed and characterized a collection of Pt prodrugs bearing amino acids as pro-moieties, linked to the Pt 4’-OH via an Nmonosubstituted carbamate ester group. The rates of hydrolysis of all compounds fell in a range suitable for their use. Derivatives with hydrophobic aminoacid side chains were notable for the high levels of Pterostilbene they regenerated in blood after intragastric administration. We selected the most promising one and we measured its levels in rat organs, following the same methodology developed for Pterostilbene. This time, the pro-drug itself was the major specie measured in all organs considered (except the brain) but, most importantly, the levels of (regenerated) Pterostilbene were drastically increased and those of its sulfate decreased in comparison with the administration of the polyphenol as such (see chapter 4).
2) As mentioned above, the intracellular processes underlying Pt benefits are still largely undefined. Several papers suggest that the induction of autophagy may contribute importantly. I decided to investigate this aspect. Autophagy is a catabolic pathway whose dysregulation is associated with various pathological conditions. As discovered by Prof. Ballabio and co-workers it is mainly controlled by transcription factor EB (TFEB), normally inhibited by mTORC1. Thus, I evaluated the effect of Pt on the autophagy master regulator. We demonstrated that this polyphenol stimulates TFEB activity by promoting its translocation to the nucleus as well as its expression. Accordingly, Pt induced an increase of LC3B protein, an autophagy marker, and an up-regulation of TFEB lysosomal target genes. These investigations have been extended to two Pt major metabolites, Pt-4’-Sulfate and DiHydroPt, as they are the main species formed upon ingestion of the natural compound. Interestingly, while the former was ineffective, the reduced form showed an activity similar to that of the parent compound but required higher concentrations. Further studies have been carried out to clarify the upstream signalling cascade. FRET-sensor based measurements have shown that Pt is able to modestly increase the concentration of cAMP, followed by the activation of CREB. This cyclic nucleotide has been previously reported to indirectly activate AMPK, a well-recognized mTORC1 antagonist. Thus, the enhancement of this cellular axis may be responsible for the activation of TFEB. Accordingly, we observed a reduction of the activity of the mammalian target of the rapamycin. However, pharmacological interventions expected to drastically increase cAMP and AMPK activity were less effective than Pt, suggesting that this phenolic compound promotes TFEB nuclear migration by modulating more than one signalling pathway. In addition to mTORC1 inhibition, the activation of TFEB is mediated by Calcineurin. Recently, it has been demonstrated that both endogenous and exogenous ROS may promote the activity of this phosphatase. We observed in in vitro studies with cultured cells that indeed Pt increased the production of these species by mitochondria and that this phenomenon is related to TFEB migration. In light of this, it is possible to speculate that this may represent an alternative way through which this phenolic compound perturbs TFEB intracellular localization (see chapter 5). The establishment of the capability of Pt to induce autophagy in cultured cells led me to test Pt as a potential therapeutic treatment for Collagen VI (ColVI) muscular dystrophies. These disorders are mainly characterized by the presence of dysfunctional mitochondria. The concomitant deficiency of the autophagic process results in the exacerbation of the pathological conditions since the apoptotic death of myofibers occurs. As known from the literature, the use of an antisense morpholino is nowadays the best strategy to obtain zebrafish animal models with a strong phenotype of ColVI-related muscle dystrophy. Therefore, we injected into fertilized eggs a designed oligonucleotide, specifically directed against the ColVI exon 9 splicing region. This results in a frame-shift deletion of the N-terminal region of the ColVIa1 triple helical domain and therefore in a strong impairment of the organization of the muscular fibers. The data I have obtained using this model indicate that Pt treatment induces a recovery of muscular structure by more than 30%, as well as a clear recovery of motor activity.This part of the project has been carried out in collaboration with Prof. Paolo Bernardi and Dr. Marco Schiavone of the University of Padua. Although these results do not provide evidence that the amelioration of the dystrophic phenotype is due to the induction of autophagy, it is credible to suppose that the same cascade of events enhanced in vitro by Pt may be relevant also in vivo. Supporting this hypothesis, Pt provoked a significant increase of a mCherry protein, whose expression is under control of cAMP responsive elements (CRE) in a zebrafish transgenic reporter (this model has been generated by Dr. Patrizia Porazzi and Prof. Natascia Tiso of the University of Padua). Further investigations need to be performed (see chapter 6). Finally, during my graduate training I have also participated in another project still ongoing in my research laboratory. The pharmacokinetic study we performed in rats showed that Pt was particularly abundant in the brain. This observation is consistent with several papers showing a role of this phenolic compound in ameliorating performance of old rodents in behavioral tests. However, also in this context, the mechanisms accounting for these effects have been poorly characterized. My in vitro data, confirmed later in zebrafish, suggest that Pt may activate CREB following an increase of cAMP. This transcription factor has been demonstrated to play a crucial role in memory consolidation as it promotes neurogenesis in the dentate gyrus, a subarea of the hippocampus, in adult individuals. Memory, among other functions, undergoes deterioration in old age. In light of this, we set up an experimental work (see chapter 7), in collaboration with Prof. Nicoletta Berardi and the research group of Dr. Alessandro Sale, aimed to evaluate the molecular changes in the dentate gyrus and the hippocampus related to the recovery of cognitive impairments in old rats by Pt, if any. The results confirmed that after chronic administration of Pt aged animal presented a remarkable improvement in memory-related behavioral tasks. Moreover, although statistics need to be improved by adding more animals, Western blot and gene expression analyses suggest that the treatment up-regulates the activity of transcription factor CREB. An increase of mitochondrial mass has been also measured. These effects were more evident in the dentate gyrus if compared to the remaining hippocampus and strongly support the occurrence of neuronal remodeling processes. However, no differences in PSD95 levels have been observed. Further markers of synaptic plasticity will need to be taken into account in future investigations. Concluding, the studies I carried out have outlined one of the mechanisms accounting for the striking properties of the Pt, characterized its organ pharmacokinetics in the rat and allowed to boost its bioavailability. Thus, they may be helpful for the development of Pt-based therapeutic/preventive treatments
Synthesis and Evaluation as Prodrugs of Hydrophilic Carbamate Ester Analogues of Resveratrol
No full textResveratrol (3,5,4'-trihydroxy-trans-stilbene) is an unfulfilled promise for health care: its exploitation is hindered by rapid conjugative metabolism in enterocytes and hepatocytes; low water solubility is a serious practical problem. To advantageously modify the physicochemical properties of the compound we have developed prodrugs in which all or part of the hydroxyl groups are linked via an N-monosubstituted carbamate ester bond to promoieties derived from glycerol or galactose, conferring higher water solubility. Kinetic studies of hydrolysis in aqueous solutions and in blood indicated that regeneration of resveratrol takes place in an appropriate time frame for delivery via oral administration. Despite their hydrophilicity some of the synthesized compounds were absorbed in the gastrointestinal tract of rats. In these cases the species found in blood after administration of a bolus consisted mainly of partially deprotected resveratrol derivatives and of the products of their glucuronidation, thus providing proof-of-principle evidence of behavior as prodrugs. The soluble compounds largely reached the lower intestinal tract. Upon administration of resveratrol, the major species found in this region was dihydroresveratrol, produced by enzymes of the intestinal flora. In experiments with a fully protected (trisubstituted) deoxygalactose containing prodrug, the major species were the prodrug itself and partially deprotected derivatives, along with small amounts of dihydroresveratrol. We conclude that the N-monosubstituted carbamate moiety is suitable for use in prodrugs of polyphenols
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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