Produzione di biomassa in vitro di Echinacea angustifolia D.C.

Le piante medicinali sono state oggetto dell’interesse dell’uomo sin dall’antichità e ancora oggi nonostante l’avvento della chimica di sintesi, la maggior parte delle medicine utilizzate deriva direttamente o indirettamente dal mondo vegetale. Infatti, molte sostanze un tempo estratte dalle piante hanno ceduto il passo a molecole di sintesi, a volte più efficaci e sicuramente più economiche da produrre. In altri casi invece, il ritorno alle molecole naturali ha determinato una rivalutazione di alcuni principi attivi di sintesi, non più efficaci o con preoccupanti effetti collaterali (Maffei, 1999). I principi attivi, i responsabili dell’attività curativa delle piante, sono prodotti del metabolismo secondario e la loro sintesi non è altro che l’attuazione di una strategia di difesa della pianta in seguito al verificarsi di condizioni avverse come fattori di stress, patogeni o predatori. La coltura in vitro di piante medicinali ha assunto grande importanza soprattutto per quelle specie, tra cui l’Echinacea angustiflolia D.C., che presentano problemi di propagazione classica o per le quali la produzione agricola non riesce a soddisfare la domanda del mercato, in quanto permette di ottenere, in maniera continua ed indipendente dalle condizioni ambientali, piante selezionate per le caratteristiche dei loro principi attivi. La specie officinale oggetto della presente tesi sperimentale è l’Echinacea angustifolia D.C., coltivata principalmente per la presenza di derivati dell’acido caffeico, alchilammidi, flavonoidi e polisaccaridi nella droga, che è costituita in prevalenza da radici e rizomi. Tali composti presentano una comprovata attività immunostimolante, antibatterica, antimicotica, antivirale, antinfiammatoria ed antiossidante. Lo scopo della seguente tesi è quello di realizzare dei sistemi di coltivazione in vitro per la produzione di biomassa di Echinacea angustifolia D.C. e di valutarne l’efficienza per poter realizzare una produzione di metaboliti secondari con metodi economicamente convenienti. In particolare, è stata messa a punto l’induzione di callo su substrati solidi in diverse condizioni ambientali caratterizzando le colture ottenute tramite la realizzazione di curve di crescita. Inoltre, poiché dalla letteratura emerge che in molti casi la produzione di metaboliti secondari è strettamente associata al grado di differenziazione dei tessuti, si è voluto indagare se colture di callo di Echinacea angustifolia D.C. in rigenerazione (ovvero masse di cellule in attiva crescita che presentano dei centri di differenziazione con apici vegetativi e primordi fogliari) fossero più idonee alla realizzazione di colture liquide per l’estrazione di principi attivi. Si è quindi avviato un sistema di coltura cellulare su substrati liquidi osservando l’andamento della crescita del callo nelle diverse condizioni testate. Tenendo in considerazione che i principi attivi presenti in questa specie sono di interesse nutraceutico, si è cercato di mettere a punto anche un sistema colturale che prevedesse l’utilizzo minimo di fitoregolatori e l’uso alternativo di sostanze naturali come il latte di cocco. Infine, la biomassa ottenuta in vitro è stata caratterizzata eseguendo un’indagine qualitativa preliminare sul contenuto in clorofilla, xantofille, carotenoidi, antocianine e flavonoli ed è stata effettuata una valutazione dell’attività antiossidante degli estratti al fine di stabilire quale dei sistemi di coltura adottati e quale dei substrati agarizzati e liquidi utilizzati, sia in grado di produrre biomassa con un elevato contenuto di sostanze d’interesse per l’industria alimentare e farmaceutica. I risultati delle prove effettuate nell’ambito di questa tesi hanno dimostrato l’attitudine di questa specie alla coltivazione in vitro. E’ stato possibile infatti, partendo da porzioni di lamina fogliare, ottenere callo attraverso l’induzione di proliferazione di cellule indifferenziate su un substrato SC (composto da: macro e micro elementi MS (Muraschige & Schoog,1962), vitamine B5 Gamborg (Gamborg, 1968), 2 mg/l BA (6-bezylaminopurine) e 2 mg/l NAA (1-naphthaleneacetic acid) al buio. L’uso del substrato Ch 0,5 BA contenente sostanze citochininiche (0,5 mg/l BA) ha indotto un processo di differenziazione nei tessuti, messo in luce prima dalla comparsa di aree pigmentate e successivamente di apici e primordi fogliari visibili. Queste colture sono cresciute attivamente soprattutto in presenza di luce dando origine ad una biomassa superiore al callo indifferenziato cresciuto sul substrato SC. Dalle curve di crescita delle colture su substrato liquido in agitazione (sistema chiuso di coltura cellulare in cui le masse di tessuto ed il substrato sono stati rinnovati ogni tre settimane) è emerso che si ha una crescita maggiore rispetto ai mezzi solidi ed in particolare l’aggiunta di latte di cocco ha indotto un’accelerazione e un aumento della crescita cellulare. I risultati ottenuti hanno quindi dimostrato che il latte di cocco può essere utilizzato come alternativa all’uso dei fitoregolatori. Dalle analisi eseguite sul materiale vegetale coltivato in vitro è emerso che il callo cresciuto sul mezzo SC, pur non mostrando alcun grado di differenziazione, ha in generale un basso contenuto di metaboliti. Invece, le biomasse rigeneranti mostrano un contenuto di flavonoli ed antocianine considerevole e sono in grado di svolgere una buona attività antiossidante. In conclusione è stato possibile realizzare dei sistemi di coltura liquida di biomasse rigeneranti che hanno mostrato buone potenzialità come strumenti per la produzione dei metaboliti secondari

PROVIDING TRANSFORMATIVE EXPERIENCES THROUGH ART FOUNDATIONS: A CONCEPTUAL MODEL FOR LUXURY FASHION BRANDS

This study aims to investigate art foundations as a potential venue for luxury fashion brands to provide a transformative experience. Providing experiences has become one of the main strategies for fashion brands to engage with their consumers. Yet, because of the proliferation of brand experiences, consumers are starting to perceive brands as not authentic anymore. Transformative experiences are believed to be more authentic and meaningful, and luxury fashion brands’ image might benefit from offering these kinds of experiences to their customers. Moreover, this is important in the perspective of strengthening the brands’ relationship with their consumers, and increasing loyalty towards the brand. According to the literature, a transformative experience is a ‘life-changing’ experience; an experience which might lead a person to gain new knowledge, increase self-awareness, perceive a sense of renewal. These kinds of experiences are considered of a more profound impact than other experiences. Hence, they might facilitate the brand in creating a deeper bond with the consumers, and strengthen consumers’ loyalty towards the brand. Art is considered a powerful facilitator in providing transformative experiences; although, in the luxury fashion context, art collaborations and brand experiences permeated with arts have become common. Thus, there are some brands which have decided to further invest in art by establishing art foundations characterised by a philanthropic scope. This study looks at these foundations as potential venue to provide transformative experiences. Based on rapid ethnography as methodology, data were collected through participant observation, two focus groups, and in-depth interviews, and were analysed utilising Template Analysis. The findings suggest that through a few implementations and improvements, the art foundation owned by the luxury fashion brand could facilitate the fashion brand in providing transformative experiences. These results have informed the creation of a conceptual framework which luxury fashion brands may implement in their foundations’ strategies to facilitate these kinds of experiences

An Attempted Suicide with Copper Sulphate injected intravenously:Pathopsysiology and Therapy about a case report

Acute copper sulphate poisoning is an unusual event, rarely following parenteral exposure, complicated by toxicological effects as haemolytic anaemia, methaemoglobinaemia , hepato-renal damage and acute rhabdomyolysis. Currently, the therapeutic management ignores unique classes of evidence and is mainly based on supportive and chelation therapies. Case details. This case report describes acute copper sulphate poisoning in a 37-year-old man who attempted suicide by self-injecting an unknown amount of copper sulphate. Within three days the patient developed severe intravascular haemolysis and rhabdomyolysis. Initial therapy relied on intensive supportive care with fluids administration, electrolyte correction and packed red blood cells transfusion. The D-penicillamine (30 mg/Kg/day per os) was prescribed as chelation therapy by the poison control centre. The N-acetilcysteine and ascorbic acid were administered to prevent further oxidative stress. Later on, two sessions of therapeutic plasma exchange were performed in order to support the drug therapy. Haemolysis and rhabdomyolysis reversed throughout the hospital stay. Discussion. In this case the antidotic and antioxidant therapy resulted effective to reverse haemolysis and rhabdomyolysis and to prevent hepatic and renal damage. Moreover, this case underlies that therapeutic plasma exchange should be considered as an additive measure to undertake since the earlier stages of the emergency intervention. Acute copper sulphate poisoning is an event complicated by toxicological effects: haemolytic anaemia, methaemoglobinaemia, hepato-renal damage, acute rhabdomyolysis. The therapeutic management ignores unique classes of evidence and is based on supportive and chelation therapies. In this case: i) the antidotic and antioxidant therapy resulted effective to reverse haemolysis and rhabdomyolysis and to prevent hepatic and renal damage, ii) therapeutic plasma exchange should be considered as an additive measure to undertake

Turin's Foodscapes: Exploring Places of Food Consumption Through the Prism of Social Practice Theory

This contribution wishes to propose an addition to the existing toolbox of techniques employed to approach and render explicit the place semantics embedded in geosocial data. Inspired by the notion of relational place introduced by human geographers, we focused on people's experience of the city derived from the aggregation of the points of view of different social groups. We analysed socio-spatial behaviour under the frame of social practice theories, defining social practices as collective social actions performed by groups of people that display a similar behaviour. Applying spatial pattern analysis and clustering on data extracted from TripAdvisor platform, we classified social groups of users depending on our prior knowledge and their spatial behaviours

Robotic total gastrectomy with intracorporeal robot-sewn anastomosis. A novel approach adopting the double-loop reconstruction method

Gastric cancer constitutes a major health problem. Robotic surgery has been progressively developed in this field. Although the feasibility of robotic procedures has been demonstrated, there are unresolved aspects being debated, including the reproducibility of intracorporeal in place of extracorporeal anastomosis. Difficulties of traditional laparoscopy have been described and there are well-known advantages of robotic systems, but few articles in literature describe a full robotic execution of the reconstructive phase while others do not give a thorough explanation how this phase was run. A new reconstructive approach, not yet described in literature, was recently adopted at our Center. Robotic total gastrectomy with D2 lymphadenectomy and a socalled ‘‘double-loop’’ reconstruction method with intracorporeal robotsewn anastomosis (Parisi’s technique) was performed in all reported cases. Preoperative, intraoperative, and postoperative data were collected and a technical note was documented. All tumors were located at the upper third of the stomach, and no conversions or intraoperative complications occurred. Histopathological analysis showed R0 resection obtained in all specimens. Hospital stay was regular in all patients and discharge was recommended starting from the 4th postoperative day. No major postoperative complications or reoperations occurred. Reconstruction of the digestive tract after total gastrectomy is one of the main areas of surgical research in the treatment of gastric cancer and in the field of minimally invasive surgery. The double-loop method is a valid simplification of the traditional technique of construction of the Roux-limb that could increase the feasibility and safety in performing a full hand-sewn intracorporeal reconstruction and it appears to fit the characteristics of the robotic system thus obtaining excellent postoperative clinical outcome

Mitochondrial Adaptations in Elderly and Young Men Skeletal Muscle Following 2 Weeks of Bed Rest and Rehabilitation

The aim of the study was to evaluate the expression levels of proteins related to mitochondrial biogenesis regulation and bioenergetics in vastus lateralis muscle biopsies from 16 elderly and 7 young people subjected to 14 days of bed-rest, causing atrophy, and subsequent 14 days of exercise training. Based on quantitative immunoblot analyses, in both groups a reduction of two key regulators of mitochondrial biogenesis/remodeling and activity, namely PGC-1alpha and Sirt3, was revealed during bedrest,with a subsequent up-regulation after rehabilitation, indicating an involvement of PGC-1alpha-Sirt3 axis in response to the treatments. A difference was observed comparing the young and elderly subjects as, for both proteins, the abundance in the elderly was more affected by immobility and less responsive to exercise. The expression levels of TOM20 and Citrate Synthase, assayed as markers of outer mitochondrial membrane and mitochondrial mass, showed a noticeable sensitivity in the elderly group, where they were affected by bed-rest and rehabilitation recalling the pattern of PGC-1alpha. TOM20 and CS remained unchanged in young subjects. Single OXPHOScomplexes showed peculiar patterns, which were in some cases dissimilar from PGC 1alpha, and suggest different influences on protein biogenesis and degradation. Overall,exercise was capable to counteract the effect of immobility, when present, except for complex V, which was markedly downregulated by bed-rest, but remained unaffected after rehabilitation, maybe as result of greater extent of degradation processes over biogenesis. Phosphorylation extent of AMPK, and its upstream activator LKB1, did not change after bed-rest and rehabilitation in either young or elderly subjects, suggestingthat the activation of energy-sensing LKB1-AMPK signaling pathway was \u201cmissed\u201d due to its transient nature, or was not triggered under our conditions. Our study demonstrates that, as far as the expression of various proteins related to mitochondrial biogenesis/remodeling, adaptations to bed-rest and rehabilitation in the two populations were different. The impact of bed-rest was greater in the elderly subjects, where the pattern (decrease after bed rest and recovery following rehabilitation) was accompanied by changes of mitochondrial mass. Modifications of protein abundance were matched with data obtained from gene expression analyses of four public human datasets focusing on related genes

The Helicobacter pylori CagY Protein Drives Gastric Th1 and Th17 Inflammation and B Cell Proliferation in Gastric MALT Lymphoma

Background: the neoplastic B cells of the Helicobacter pylori-related low-grade gastric mucosa-associated lymphoid tissue (MALT) lymphoma proliferate in response to H. pylori, however, the nature of the H. pylori antigen responsible for proliferation is still unknown. The purpose of the study was to dissect whether CagY might be the H. pylori antigen able to drive B cell proliferation. Methods: the B cells and the clonal progeny of T cells from the gastric mucosa of five patients with MALT lymphoma were compared with those of T cell clones obtained from five H. pylori-infected patients with chronic gastritis. The T cell clones were assessed for their specificity to H. pylori CagY, cytokine profile and helper function for B cell proliferation. Results: 22 of 158 CD4(+) (13.9%) gastric clones from MALT lymphoma and three of 179 CD4(+) (1.7%) clones from chronic gastritis recognized CagY. CagY predominantly drives Interferon-gamma (IFN-gamma) and Interleukin-17 (IL-17) secretion by gastric CD4(+) T cells from H. pylori-infected patients with low-grade gastric MALT lymphoma. All MALT lymphoma-derived clones dose dependently increased their B cell help, whereas clones from chronic gastritis lost helper activity at T-to-B-cell ratios greater than 1. Conclusion: the results obtained indicate that CagY drives both B cell proliferation and T cell activation in gastric MALT lymphomas

Thrombosis in vasculitis: from pathogenesis to treatment

In recent years, the relationship between inflammation and thrombosis has been deeply investigated and it is now clear that immune and coagulation systems are functionally interconnected. Inflammation-induced thrombosis is by now considered a feature not only of autoimmune rheumatic diseases, but also of systemic vasculitides such as Behçet’s syndrome, ANCA-associated vasculitis or giant cells arteritis, especially during active disease. These findings have important consequences in terms of management and treatment. Indeed, Behçet’syndrome requires immunosuppressive agents for vascular involvement rather than anticoagulation or antiplatelet therapy, and it is conceivable that also in ANCA-associated vasculitis or large vessel-vasculitis an aggressive anti-inflammatory treatment during active disease could reduce the risk of thrombotic events in early stages. In this review we discuss thrombosis in vasculitides, especially in Behçet’s syndrome, ANCA-associated vasculitis and large-vessel vasculitis, and provide pathogenetic and clinical clues for the different specialists involved in the care of these patients