Correlation between insulin resistance and treatment-resistant acne

Physiologically during puberty and adolescence, when juvenile acne usually appears, the response to a glucose load is increased if compared to the one observed in adult and at pre-pubertal age, while insulin sensitivity is reduced. Insulin is a hormone that acts at different levels along the axis which controls the sex hormones. It increases the release of LH and FSH by pituitary gland, stimulates the synthesis of androgens in the gonads and stimulates the synthesis of androgenic precursors in adrenal glands. Finally, it acts in the liver by inhibiting the synthesis of Sex Hormone Binding Globulin (SHBG). Insulin is also able to act directly on the production of sebum and amplify the effects of Iinsulin Growth Factor-1 in the skin, inhibiting the synthesis of its binding protein (IGF Binding Protein-1). In female subjects with acne and Polycystic Ovary Syndrome (PCOS) insulin resistance is a well known pathogenetic factor, while the relationship between acne and insulin resistance has been poorly investigated in males so far. The purpose of this study is to investigate the correlation between insulin resistance and acne in young males who do not respond to common therapies. Clinical and biochemical parameters of glucose, lipid metabolism, androgens and IGF-1 were evaluated. Insulin resistance was estimated by Homeostasis Model assessment (HOMA-IR) and Oral Glucose Tolerance Test was also performed. We found that subjects with acne had higher Sistolic and Diastolic Blood Pressure, Waist/Hip Ratio, Waist Circumference, 120' OGTT serum insulin and serum IGF-1 and lower HDL-cholesterol than subjects of comparable age and gender without acne. The results thus obtained confirmed what other authors have recently reported about a metabolic imbalance in young males with acne. Furthermore, these results support the hypothesis that insulin resistance might play an important role in the pathogenesis of treatment-resistant acne in males.

Da Laodice I a Laodice III: l'orizzonte politico delle regine seleucidi

Questo studio indaga la tradizione storiografica sulle regine seleucidi di III secolo a.C. - da Laodice I fino all’ascesa al trono di Laodice III - alla luce delle testimonianze documentarie. L’intento è stabilire l’orizzonte politico dell’azione delle regine nel momento meno conosciuto e più buio della storia del regno di Siria. Il III secolo seleucidico è infatti un periodo caratterizzato da incertezza nella ricostruzione evenemenziale e nell’analisi delle dinamiche sociopolitiche, soprattutto a causa dell’interesse relativo e limitato delle fonti storiografiche antiche a noi giunte relativamente alle vicende della monarchia seleucidica precedenti l’avvento di Antioco III il Grande. In questo panorama rappresenta un’eccezione il caso di Laodice I, che è al centro di un rilevante numero di testimonianze. Tenendo conto della differente natura delle fonti e della complessità del contesto evenemenziale, non sempre ricostruibile, è stata studiata la figura di Laodice I nell’intento di comprendere il paradigma di regalità femminile stabilito dalle azioni della regina, e quindi l’influenza di tale paradigma sulla generazione seleucide successiva. La ricerca si articola in tre sezioni, legate agli eventi principali del III secolo seleucide che vedono coinvolta la regina. La prima parte è dedicata all’attività di Laodice I dopo il secondo matrimonio di Antioco II con la tolemaica Berenice. La seconda parte riguarda il ruolo di Laodice nei complessi eventi che seguirono la morte del marito Antioco II nel 246 e nella Terza Guerra Siriaca, che vide la regina a fianco del figlio Seleuco II contrapporsi a Tolemeo III e Berenice Sira. La terza sezione si occupa dell’azione di Laodice I nella Guerra Fraterna al fianco di Antioco Ierace contro Seleuco II e Laodice II. Nella conclusione si riflette sull’influenza delle azioni politiche di Laodice I sulla scelta di Antioco III di sposare la cugina Laodice III, prima principessa del Ponto entrata nella dinastia regnante.

Metagenomic trajectory of gut microbiome in the human lifespan

Co-evolving with the human host, gut microbiota establishes configurations, which vary under the pressure of inflammation, disease, ageing, diet and lifestyle. In order to describe the multi-stability of the microbiome-host relationship, we studied specific tracts of the bacterial trajectory during the human lifespan and we characterized peculiar deviations from the hypothetical development, caused by disease, using molecular techniques, such as phylogenetic microarray and next-generation sequencing. Firstly, we characterized the enterocyte-associated microbiota in breast-fed infants and adults, describing remarkable differences between the two groups of subjects. Successively, we investigated the impact of atopy on the development of the microbiome in Italian childrens, highlithing conspicuous deviations from the child-type microbiota of the Italian controls. To explore variation in the gut microbiota depending on geographical origins, which reflect different lifestyles, we compared the phylogenetic diversity of the intestinal microbiota of the Hadza hunter-gatherers of Tanzania and Italian adults. Additionally, we characterized the aged-type microbiome, describing the changes occurred in the metabolic potential of the gut microbiota of centenarians with respect to younger individuals, as a part of the pathophysiolology of the ageing process. Finally, we evaluated the impact of a probiotics intervention on the intestinal microbiota of elderly people, showing the repair of some age-related dysbioses. These studies contribute to elucidate several aspects of the intestinal microbiome development during the human lifespan, depicting the microbiota as an extremely plastic entity, capable of being reconfigured in response to different environmental factors and/or stressors of endogenous origin.

Filologia Classica: dal segno, al suono, al significato

Il lavoro presenta le ricerche svolte ed i risultati ottenuti per consentire alle persone non vedenti lo studio quanto più autonomo del greco antico tramite sintesi vocale. Una introduzione storica ripercorre la prima vicenda professionale dell'esercizio della professione docente delle lettere classiche da parte di un soggetto privo di vista, mentre il riferimento alla legislazione contemporanea tenta di disegnare una cornice capace dei più moderni approdi supportati dalle tecnologie informatiche. La Tesi illustra infine il funzionamento dei vari dispositivi prodotti, mentre una importante appendice ripercorre le pubblicazioni via via curate dal Gruppo di Studio, accogliendo infine le trascrizioni dei codici di programmazione delle sintesi vocali realizzate. Essa affida così all'Università la prosecuzione delle ricerche delineando ulteriori ipotesi di sviluppo.

Moduli consensuali nei rapporti tra Stato e autonomie territoriali e ruolo delle Conferenze

Ad oltre un decennio dalla riforma costituzionale del 2001, la non compiuta attuazione della stessa sembra condurre ad una nuova ed assai prossima revisione costituzionale. Nel mutato quadro costituzionale di quest’ultimo periodo l’attenzione e l’interesse sono stati richiamati da una grande quantità di accordi, di intese e di altri moduli consensuali, introdotti ed esitati nei rapporti tra Stato ed autonomie territoriali. Dall’esame del sistema delle Conferenze si è evidenziata la loro indispensabilità ai fini del coordinamento delle azioni politico –amministrative delle autonomie territoriali, ma è anche venuta fuori l’esigenza di ricorrere frequentemente ad altri tipi di moduli consensuali, spesso non tipizzati in ambito legislativo. I principi di sussidiarietà e di leale collaborazione inducono non di rado ad assumere, nella concretezza, forme eterogenee poco chiare e confuse (accordo, intesa, concerto, parere) che, pur considerati i contributi offerti dalla dottrina e dalla giurisprudenza, richiederebbero un intervento da parte del legislatore. L’approfondimento tematico ha rilevato, nel contesto di rapporti tra Stato ed autonomie territoriali, un movimento per così dire ondulatorio tra accese spinte autonomistiche e rimarcate esigenze centralistiche, lasciando ancora nella prospettiva una operativa ed efficiente armonia istituzionale.

Advanced aspects of radiation protection in the use of particle accelerators in the medical field

In this work, the well-known MC code FLUKA was used to simulate the GE PETrace cyclotron (16.5 MeV) installed at “S. Orsola-Malpighi” University Hospital (Bologna, IT) and routinely used in the production of positron emitting radionuclides. Simulations yielded estimates of various quantities of interest, including: the effective dose distribution around the equipment; the effective number of neutron produced per incident proton and their spectral distribution; the activation of the structure of the cyclotron and the vault walls; the activation of the ambient air, in particular the production of 41Ar, the assessment of the saturation yield of radionuclides used in nuclear medicine. The simulations were validated against experimental measurements in terms of physical and transport parameters to be used at the energy range of interest in the medical field. The validated model was also extensively used in several practical applications uncluding the direct cyclotron production of non-standard radionuclides such as 99mTc, the production of medical radionuclides at TRIUMF (Vancouver, CA) TR13 cyclotron (13 MeV), the complete design of the new PET facility of “Sacro Cuore – Don Calabria” Hospital (Negrar, IT), including the ACSI TR19 (19 MeV) cyclotron, the dose field around the energy selection system (degrader) of a proton therapy cyclotron, the design of plug-doors for a new cyclotron facility, in which a 70 MeV cyclotron will be installed, and the partial decommissioning of a PET facility, including the replacement of a Scanditronix MC17 cyclotron with a new TR19 cyclotron.

I "governi del Presidente"

Gli avvenimenti politico-istituzionali degli ultimi anni hanno determinato un vivace dibattito sulla questione dei cc.dd. “governi del Presidente”, con cui si suole far riferimento ad Esecutivi il cui procedimento di formazione e le cui crisi registrano un interventismo del Presidente della Repubblica superiore a quello richiesto e/o consentito dal dettato costituzionale. Obiettivo dell’elaborato è duplice: fornire un tentativo definitorio della categoria “governi del Presidente”, nonché valutare la compatibilità di questa con la forma di governo parlamentare, soprattutto in considerazione dei recenti sviluppi delle dinamiche politico-istituzionali italiane. Sulla base di questo presupposto, nel presente lavoro si procede a una ricostruzione dell’evoluzione della prassi e delle consuetudini costituzionali in merito all’esercizio dei poteri presidenziali nella gestione delle crisi di governo, con particolare riferimento al procedimento di formazione dell’Esecutivo. La tesi si presenta articolata in due parti, corrispondenti a due differenti periodi storici (cc.dd. Prima e Seconda Repubblica), organizzate per capitoli, relativi alle principali fasi di evoluzione del sistema politico attraversate nel corso della storia della Repubblica. Questa scelta si giustifica in ragione del fatto che il modus operandi del Presidente della Repubblica risulta fortemente condizionato dai mutamenti del contesto politico-istituzionale. Nei singoli paragrafi, dedicati ai presidenti, vengono trattati l’elezione del Presidente, le modalità di formazione dei governi nominati, nonché ulteriori temi la cui disamina è stata ritenuta opportuna allo scopo di fornire una contestualizzazione più adeguata dell’analisi svolta (utilizzo del potere di esternazione e di scioglimento delle Camere, gestione delle attività di politica interna e politica estera, principali avvenimenti dei periodi storici considerati, e così via).

Microbial ecology of biotechnological processes

The investigation of phylogenetic diversity and functionality of complex microbial communities in relation to changes in the environmental conditions represents a major challenge of microbial ecology research. Nowadays, particular attention is paid to microbial communities occurring at environmental sites contaminated by recalcitrant and toxic organic compounds. Extended research has evidenced that such communities evolve some metabolic abilities leading to the partial degradation or complete mineralization of the contaminants. Determination of such biodegradation potential can be the starting point for the development of cost effective biotechnological processes for the bioremediation of contaminated matrices. This work showed how metagenomics-based microbial ecology investigations supported the choice or the development of three different bioremediation strategies. First, PCR-DGGE and PCR-cloning approaches served the molecular characterization of microbial communities enriched through sequential development stages of an aerobic cometabolic process for the treatment of groundwater contaminated by chlorinated aliphatic hydrocarbons inside an immobilized-biomass packed bed bioreactor (PBR). In this case the analyses revealed homogeneous growth and structure of immobilized communities throughout the PBR and the occurrence of dominant microbial phylotypes of the genera Rhodococcus, Comamonas and Acidovorax, which probably drive the biodegradation process. The same molecular approaches were employed to characterize sludge microbial communities selected and enriched during the treatment of municipal wastewater coupled with the production of polyhydroxyalkanoates (PHA). Known PHA-accumulating microorganisms identified were affiliated with the genera Zooglea, Acidovorax and Hydrogenophaga. Finally, the molecular investigation concerned communities of polycyclic aromatic hydrocarbon (PAH) contaminated soil subjected to rhizoremediation with willow roots or fertilization-based treatments. The metabolic ability to biodegrade naphthalene, as a representative model for PAH, was assessed by means of stable isotope probing in combination with high-throughput sequencing analysis. The phylogenetic diversity of microbial populations able to derive carbon from naphthalene was evaluated as a function of the type of treatment.

Bioremediation of PCB-contaminated marine sediments: From identification of indigenous dehalorespirers to enhancement of microbial reductive dechlorination

Marine sediments are the main accumulation reservoir of organic recalcitrant pollutants such as polychlorinated biphenyls (PCBs). In the anoxic conditions typical of these sediments, anaerobic bacteria of the phylum Chloroflexi are able to attack these compounds in a process called microbial reductive dechlorination. Such activity and members of this phylum were detected in PCB-impacted sediments of the Venice Lagoon. The aim of this work was to investigate microbial reductive dechlorination and design bioremediation approaches for marine sediments of the area. Three out of six sediment cultures from different sampling areas exhibited dechlorination activities in the same conditions of the site and two phylotypes (VLD-1 and VLD-2) were detected and correlated to this metabolism. Biostimulation was tested on enriched dechlorinating sediment cultures from the same site using five different electron donors, of which lactate was the best biostimulating agent; complementation of microbial and chemical dechlorination catalyzed by biogenic zerovalent Pd nanoparticles was not effective due to sulfide poisoning of the catalyst. A new biosurfactant-producing strain of Shewanella frigidimarina was concomitantly obtained from hydrocarbon-degrading marine cultures and selected because of the low toxicity of its product. All these findings were then exploited to develop bioremediation lab-scale tests in shaken reactors and static microcosms on real sediments and water of the Venice lagoon, testing i) a bioaugmentation approach, with a selected enriched sediment culture from the same area, ii) a biostimulation approach with lactate as electron donor, iii) a bioavailability enhancement with the supplementation of the newly-discovered biosurfactant, and iv) all possible combinations of the afore-mentioned approaches. The best bioremediation approach resulted to be a combination of bioaugmentation and bioremediation and it could be a starting point to design bioremediation process for actual marine sediments of the Venice Lagoon area.

Sviluppo di metodologie innovative di supporto decisionale alla progettazione basate su indicatori di prestazione

Il progetto di ricerca è finalizzato allo sviluppo di una metodologia innovativa di supporto decisionale nel processo di selezione tra alternative progettuali, basata su indicatori di prestazione. In particolare il lavoro si è focalizzato sulla definizione d’indicatori atti a supportare la decisione negli interventi di sbottigliamento di un impianto di processo. Sono stati sviluppati due indicatori, “bottleneck indicators”, che permettono di valutare la reale necessità dello sbottigliamento, individuando le cause che impediscono la produzione e lo sfruttamento delle apparecchiature. Questi sono stati validati attraverso l’applicazione all’analisi di un intervento su un impianto esistente e verificando che lo sfruttamento delle apparecchiature fosse correttamente individuato. Definita la necessità dell’intervento di sbottigliamento, è stato affrontato il problema della selezione tra alternative di processo possibili per realizzarlo. È stato applicato alla scelta un metodo basato su indicatori di sostenibilità che consente di confrontare le alternative considerando non solo il ritorno economico degli investimenti ma anche gli impatti su ambiente e sicurezza, e che è stato ulteriormente sviluppato in questa tesi. Sono stati definiti due indicatori, “area hazard indicators”, relativi alle emissioni fuggitive, per integrare questi aspetti nell’analisi della sostenibilità delle alternative. Per migliorare l’accuratezza nella quantificazione degli impatti è stato sviluppato un nuovo modello previsionale atto alla stima delle emissioni fuggitive di un impianto, basato unicamente sui dati disponibili in fase progettuale, che tiene conto delle tipologie di sorgenti emettitrici, dei loro meccanismi di perdita e della manutenzione. Validato mediante il confronto con dati sperimentali di un impianto produttivo, si è dimostrato che tale metodo è indispensabile per un corretto confronto delle alternative poiché i modelli esistenti sovrastimano eccessivamente le emissioni reali. Infine applicando gli indicatori ad un impianto esistente si è dimostrato che sono fondamentali per semplificare il processo decisionale, fornendo chiare e precise indicazioni impiegando un numero limitato di informazioni per ricavarle.

Il diritto alla mobilita' fra dimensione europea, costituzioni statali e autonomie territoriali

La tesi si prefigge l'obbiettivo di offrire una ricostruzione logico sistematica della disciplina giuridica che regola i trasporti pubblici locali in ambito regionale, statale e comunitario, affrontando le principali questioni interpretative e di coordinamento che esse pongono. Nella primo capitolo, viene analizzato l'evoluzione storica della normativa nazionale che regola il trasporto pubblico locale, soffermandosi soprattutto sulla riforma del trasporto pubblico locale introdotte dal d.lgs. 422/1997. Particolare attenzione è stata posta agli aspetti di programmazione e finanziamento nonché alle modalità di gestione del trasporto pubblico locale, in quanto il quadro normativo applicabile è caratterizzato da un’estrema complessità dovuta ai numerosi interventi legislativi. Nel secondo capitolo viene esaminato l'evoluzione dell'intervento comunitario in materi di trasporto pubblico locale, partendo dal (CE) n. 1191/69 che si limitava a disciplinare gli aiuti di Stato, fino alla normativa quadro per il settore (Regolamento (CE) n. 1370/2007). L'obbiettivo è quello di verificare se le scelte del legislatore italiano, per quanto concerne le modalità di gestione del trasporto pubblico locale possano dirsi coerenti con le scelte a livello comunitario previste dal Regolamento (CE) n. 1370/2007. Viene inoltre affronta la questione dell'articolazione della potestà normativa e amministrativa del settore dei trasporti pubblici locali nelle disposizioni del Titolo V della Costituzione. Lo studio si sofferma soprattutto sulla giurisprudenza della Corte costituzionale per tracciare una chiara individuazione del riparto delle competenze tra Stato e Regioni in materia. Infine nell'ultima parte, esamina le diverse problematiche interpretative e applicative della normativa che disciplina il settore del TPL, dovute all'azzeramento della normativa generale dei servizi pubblici locali di rilevanza economica in seguito al referendum abrogativo del 12 e 13 giugno 2011, nonché della illegittimità costituzionale della normativa contenuta nell'art 4 del d.l. n. 138/2011, ad opera della sentenza della Corte costituzionale n. 199/2012.

La prova penale nelle indagini difensive. Analisi comparata degli strumenti processuali concessi alla difesa nei sistemi italiano e statunitense.

L’elaborato si occupa di fare il punto in materia di indagini difensive a tre lustri dall’entrata in vigore della legge n. 397/2000, epilogo di un lungo processo evolutivo che ha visto da un lato, una gestazione faticosa e travagliata, dall’altro, un prodotto normativo accolto dagli operatori in un contesto di scetticismo generale. In un panorama normativo e giurisprudenziale in continua evoluzione, i paradigmi dettati dagli artt. 24 e 111 della Costituzione, in tema di diritto alla difesa e di formazione della prova penale secondo il principio del contraddittorio tra le parti, in condizioni di parità, richiedono che il sistema giustizia offra sia all’indagato che all’imputato sufficienti strumenti difensivi. Tenuto conto delle diversità che caratterizzano naturalmente i ruoli dell’accusa e della difesa che impongono asimmetrie genetiche inevitabili, l’obiettivo della ricerca consiste nella disamina degli strumenti idonei a garantire il diritto alla prova della difesa in ogni stato e grado del procedimento, nel tentativo di realizzare compiutamente il principio di parità accusa - difesa nel processo penale. La ricerca si dipana attraverso tre direttrici: l’analisi dello statuto sulle investigazioni difensive nella sua evoluzione storica sino ai giorni nostri, lo studio della prova penale nel sistema americano e, infine, in alcune considerazioni finali espresse in chiave comparatistica. Le suggestioni proposte sono caratterizzate da un denominatore comune, ovvero dal presupposto che per contraddire è necessario conoscere e che solo per tale via sia possibile, finalmente, riconoscere il diritto di difendersi indagando.

Impatto di rifaximina sul microbiota intestinale: selezione di bifidobatteri antibiotico resistenti

The ideal approach for the long term treatment of intestinal disorders, such as inflammatory bowel disease (IBD), is represented by a safe and well tolerated therapy able to reduce mucosal inflammation and maintain homeostasis of the intestinal microbiota. A combined therapy with antimicrobial agents, to reduce antigenic load, and immunomodulators, to ameliorate the dysregulated responses, followed by probiotic supplementation has been proposed. Because of the complementary mechanisms of action of antibiotics and probiotics, a combined therapeutic approach would give advantages in terms of enlargement of the antimicrobial spectrum, due to the barrier effect of probiotic bacteria, and limitation of some side effects of traditional chemiotherapy (i.e. indiscriminate decrease of aggressive and protective intestinal bacteria, altered absorption of nutrient elements, allergic and inflammatory reactions). Rifaximin (4-deoxy-4’-methylpyrido[1’,2’-1,2]imidazo[5,4-c]rifamycin SV) is a product of synthesis experiments designed to modify the parent compound, rifamycin, in order to achieve low gastrointestinal absorption while retaining good antibacterial activity. Both experimental and clinical pharmacology clearly show that this compound is a non systemic antibiotic with a broad spectrum of antibacterial action, covering Gram-positive and Gram-negative organisms, both aerobes and anaerobes. Being virtually non absorbed, its bioavailability within the gastrointestinal tract is rather high with intraluminal and faecal drug concentrations that largely exceed the MIC values observed in vitro against a wide range of pathogenic microorganisms. The gastrointestinal tract represents therefore the primary therapeutic target and gastrointestinal infections the main indication. The little value of rifaximin outside the enteric area minimizes both antimicrobial resistance and systemic adverse events. Fermented dairy products enriched with probiotic bacteria have developed into one of the most successful categories of functional foods. Probiotics are defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” (FAO/WHO, 2002), and mainly include Lactobacillus and Bifidobacterium species. Probiotic bacteria exert a direct effect on the intestinal microbiota of the host and contribute to organoleptic, rheological and nutritional properties of food. Administration of pharmaceutical probiotic formula has been associated with therapeutic effects in treatment of diarrhoea, constipation, flatulence, enteropathogens colonization, gastroenteritis, hypercholesterolemia, IBD, such as ulcerative colitis (UC), Crohn’s disease, pouchitis and irritable bowel syndrome. Prerequisites for probiotics are to be effective and safe. The characteristics of an effective probiotic for gastrointestinal tract disorders are tolerance to upper gastrointestinal environment (resistance to digestion by enteric or pancreatic enzymes, gastric acid and bile), adhesion on intestinal surface to lengthen the retention time, ability to prevent the adherence, establishment and/or replication of pathogens, production of antimicrobial substances, degradation of toxic catabolites by bacterial detoxifying enzymatic activities, and modulation of the host immune responses. This study was carried out using a validated three-stage fermentative continuous system and it is aimed to investigate the effect of rifaximin on the colonic microbial flora of a healthy individual, in terms of bacterial composition and production of fermentative metabolic end products. Moreover, this is the first study that investigates in vitro the impact of the simultaneous administration of the antibiotic rifaximin and the probiotic B. lactis BI07 on the intestinal microbiota. Bacterial groups of interest were evaluated using culture-based methods and molecular culture-independent techniques (FISH, PCR-DGGE). Metabolic outputs in terms of SCFA profiles were determined by HPLC analysis. Collected data demonstrated that rifaximin as well as antibiotic and probiotic treatment did not change drastically the intestinal microflora, whereas bacteria belonging to Bifidobacterium and Lactobacillus significantly increase over the course of the treatment, suggesting a spontaneous upsurge of rifaximin resistance. These results are in agreement with a previous study, in which it has been demonstrated that rifaximin administration in patients with UC, affects the host with minor variations of the intestinal microflora, and that the microbiota is restored over a wash-out period. In particular, several Bifidobacterium rifaximin resistant mutants could be isolated during the antibiotic treatment, but they disappeared after the antibiotic suspension. Furthermore, bacteria belonging to Atopobium spp. and E. rectale/Clostridium cluster XIVa increased significantly after rifaximin and probiotic treatment. Atopobium genus and E. rectale/Clostridium cluster XIVa are saccharolytic, butyrate-producing bacteria, and for these characteristics they are widely considered health-promoting microorganisms. The absence of major variations in the intestinal microflora of a healthy individual and the significant increase in probiotic and health-promoting bacteria concentrations support the rationale of the administration of rifaximin as efficacious and non-dysbiosis promoting therapy and suggest the efficacy of an antibiotic/probiotic combined treatment in several gut pathologies, such as IBD. To assess the use of an antibiotic/probiotic combination for clinical management of intestinal disorders, genetic, proteomic and physiologic approaches were employed to elucidate molecular mechanisms determining rifaximin resistance in Bifidobacterium, and the expected interactions occurring in the gut between these bacteria and the drug. The ability of an antimicrobial agent to select resistance is a relevant factor that affects its usefulness and may diminish its useful life. Rifaximin resistance phenotype was easily acquired by all bifidobacteria analyzed [type strains of the most representative intestinal bifidobacterial species (B. infantis, B. breve, B. longum, B. adolescentis and B. bifidum) and three bifidobacteria included in a pharmaceutical probiotic preparation (B. lactis BI07, B. breve BBSF and B. longum BL04)] and persisted for more than 400 bacterial generations in the absence of selective pressure. Exclusion of any reversion phenomenon suggested two hypotheses: (i) stable and immobile genetic elements encode resistance; (ii) the drug moiety does not act as an inducer of the resistance phenotype, but enables selection of resistant mutants. Since point mutations in rpoB have been indicated as representing the principal factor determining rifampicin resistance in E. coli and M. tuberculosis, whether a similar mechanism also occurs in Bifidobacterium was verified. The analysis of a 129 bp rpoB core region of several wild-type and resistant bifidobacteria revealed five different types of miss-sense mutations in codons 513, 516, 522 and 529. Position 529 was a novel mutation site, not previously described, and position 522 appeared interesting for both the double point substitutions and the heterogeneous profile of nucleotide changes. The sequence heterogeneity of codon 522 in Bifidobacterium leads to hypothesize an indirect role of its encoded amino acid in the binding with the rifaximin moiety. These results demonstrated the chromosomal nature of rifaximin resistance in Bifidobacterium, minimizing risk factors for horizontal transmission of resistance elements between intestinal microbial species. Further proteomic and physiologic investigations were carried out using B. lactis BI07, component of a pharmaceutical probiotic preparation, as a model strain. The choice of this strain was determined based on the following elements: (i) B. lactis BI07 is able to survive and persist in the gut; (ii) a proteomic overview of this strain has been recently reported. The involvement of metabolic changes associated with rifaximin resistance was investigated by proteomic analysis performed with two-dimensional electrophoresis and mass spectrometry. Comparative proteomic mapping of BI07-wt and BI07-res revealed that most differences in protein expression patterns were genetically encoded rather than induced by antibiotic exposure. In particular, rifaximin resistance phenotype was characterized by increased expression levels of stress proteins. Overexpression of stress proteins was expected, as they represent a common non specific response by bacteria when stimulated by different shock conditions, including exposure to toxic agents like heavy metals, oxidants, acids, bile salts and antibiotics. Also, positive transcription regulators were found to be overexpressed in BI07-res, suggesting that bacteria could activate compensatory mechanisms to assist the transcription process in the presence of RNA polymerase inhibitors. Other differences in expression profiles were related to proteins involved in central metabolism; these modifications suggest metabolic disadvantages of resistant mutants in comparison with sensitive bifidobacteria in the gut environment, without selective pressure, explaining their disappearance from faeces of patients with UC after interruption of antibiotic treatment. The differences observed between BI07-wt e BI07-res proteomic patterns, as well as the high frequency of silent mutations reported for resistant mutants of Bifidobacterium could be the consequences of an increased mutation rate, mechanism which may lead to persistence of resistant bacteria in the population. However, the in vivo disappearance of resistant mutants in absence of selective pressure, allows excluding the upsurge of compensatory mutations without loss of resistance. Furthermore, the proteomic characterization of the resistant phenotype suggests that rifaximin resistance is associated with a reduced bacterial fitness in B. lactis BI07-res, supporting the hypothesis of a biological cost of antibiotic resistance in Bifidobacterium. The hypothesis of rifaximin inactivation by bacterial enzymatic activities was verified by using liquid chromatography coupled with tandem mass spectrometry. Neither chemical modifications nor degradation derivatives of the rifaximin moiety were detected. The exclusion of a biodegradation pattern for the drug was further supported by the quantitative recovery in BI07-res culture fractions of the total rifaximin amount (100 μg/ml) added to the culture medium. To confirm the main role of the mutation on the β chain of RNA polymerase in rifaximin resistance acquisition, transcription activity of crude enzymatic extracts of BI07-res cells was evaluated. Although the inhibition effects of rifaximin on in vitro transcription were definitely higher for BI07-wt than for BI07-res, a partial resistance of the mutated RNA polymerase at rifaximin concentrations > 10 μg/ml was supposed, on the basis of the calculated differences in inhibition percentages between BI07-wt and BI07-res. By considering the resistance of entire BI07-res cells to rifaximin concentrations > 100 μg/ml, supplementary resistance mechanisms may take place in vivo. A barrier for the rifaximin uptake in BI07-res cells was suggested in this study, on the basis of the major portion of the antibiotic found to be bound to the cellular pellet respect to the portion recovered in the cellular lysate. Related to this finding, a resistance mechanism involving changes of membrane permeability was supposed. A previous study supports this hypothesis, demonstrating the involvement of surface properties and permeability in natural resistance to rifampicin in mycobacteria, isolated from cases of human infection, which possessed a rifampicin-susceptible RNA polymerase. To understand the mechanism of membrane barrier, variations in percentage of saturated and unsaturated FAs and their methylation products in BI07-wt and BI07-res membranes were investigated. While saturated FAs confer rigidity to membrane and resistance to stress agents, such as antibiotics, a high level of lipid unsaturation is associated with high fluidity and susceptibility to stresses. Thus, the higher percentage of saturated FAs during the stationary phase of BI07-res could represent a defence mechanism of mutant cells to prevent the antibiotic uptake. Furthermore, the increase of CFAs such as dihydrosterculic acid during the stationary phase of BI07-res suggests that this CFA could be more suitable than its isomer lactobacillic acid to interact with and prevent the penetration of exogenous molecules including rifaximin. Finally, the impact of rifaximin on immune regulatory functions of the gut was evaluated. It has been suggested a potential anti-inflammatory effect of rifaximin, with reduced secretion of IFN-γ in a rodent model of colitis. Analogously, it has been reported a significant decrease in IL-8, MCP-1, MCP-3 e IL-10 levels in patients affected by pouchitis, treated with a combined therapy of rifaximin and ciprofloxacin. Since rifaximin enables in vivo and in vitro selection of Bifidobacterium resistant mutants with high frequency, the immunomodulation activities of rifaximin associated with a B. lactis resistant mutant were also taken into account. Data obtained from PBMC stimulation experiments suggest the following conclusions: (i) rifaximin does not exert any effect on production of IL-1β, IL-6 and IL-10, whereas it weakly stimulates production of TNF-α; (ii) B. lactis appears as a good inducer of IL-1β, IL-6 and TNF-α; (iii) combination of BI07-res and rifaximin exhibits a lower stimulation effect than BI07-res alone, especially for IL-6. These results confirm the potential anti-inflammatory effect of rifaximin, and are in agreement with several studies that report a transient pro-inflammatory response associated with probiotic administration. The understanding of the molecular factors determining rifaximin resistance in the genus Bifidobacterium assumes an applicative significance at pharmaceutical and medical level, as it represents the scientific basis to justify the simultaneous use of the antibiotic rifaximin and probiotic bifidobacteria in the clinical treatment of intestinal disorders.

Processi di biorefining per l'estrazione di secondary chemical building blocks da sottoprotti dell'agro-industria

Phenol and cresols represent a good example of primary chemical building blocks of which 2.8 million tons are currently produced in Europe each year. Currently, these primary phenolic building blocks are produced by refining processes from fossil hydrocarbons: 5% of the world-wide production comes from coal (which contains 0.2% of phenols) through the distillation of the tar residue after the production of coke, while 95% of current world production of phenol is produced by the distillation and cracking of crude oil. In nature phenolic compounds are present in terrestrial higher plants and ferns in several different chemical structures while they are essentially absent in lower organisms and in animals. Biomass (which contain 3-8% of phenols) represents a substantial source of secondary chemical building blocks presently underexploited. These phenolic derivatives are currently used in tens thousand of tons to produce high cost products such as food additives and flavours (i.e. vanillin), fine chemicals (i.e. non-steroidal anti-inflammatory drugs such as ibuprofen or flurbiprofen) and polymers (i.e. poly p-vinylphenol, a photosensitive polymer for electronic and optoelectronic applications). European agrifood waste represents a low cost abundant raw material (250 millions tons per year) which does not subtract land use and processing resources from necessary sustainable food production. The class of phenolic compounds is essentially constituted by simple phenols, phenolic acids, hydroxycinnamic acid derivatives, flavonoids and lignans. As in the case of coke production, the removal of the phenolic contents from biomass upgrades also the residual biomass. Focusing on the phenolic component of agrifood wastes, huge processing and marketing opportunities open since phenols are used as chemical intermediates for a large number of applications, ranging from pharmaceuticals, agricultural chemicals, food ingredients etc. Following this approach we developed a biorefining process to recover the phenolic fraction of wheat bran based on enzymatic commercial biocatalysts in completely water based process, and polymeric resins with the aim of substituting secondary chemical building blocks with the same compounds naturally present in biomass. We characterized several industrial enzymatic product for their ability to hydrolize the different molecular features that are present in wheat bran cell walls structures, focusing on the hydrolysis of polysaccharidic chains and phenolics cross links. This industrial biocatalysts were tested on wheat bran and the optimized process allowed to liquefy up to the 60 % of the treated matter. The enzymatic treatment was also able to solubilise up to the 30 % of the alkali extractable ferulic acid. An extraction process of the phenolic fraction of the hydrolyzed wheat bran based on an adsorbtion/desorption process on styrene-polyvinyl benzene weak cation-exchange resin Amberlite IRA 95 was developed. The efficiency of the resin was tested on different model system containing ferulic acid and the adsorption and desorption working parameters optimized for the crude enzymatic hydrolyzed wheat bran. The extraction process developed had an overall yield of the 82% and allowed to obtain concentrated extracts containing up to 3000 ppm of ferulic acid. The crude enzymatic hydrolyzed wheat bran and the concentrated extract were finally used as substrate in a bioconversion process of ferulic acid into vanillin through resting cells fermentation. The bioconversion process had a yields in vanillin of 60-70% within 5-6 hours of fermentation. Our findings are the first step on the way to demonstrating the economical feasibility for the recovery of biophenols from agrifood wastes through a whole crop approach in a sustainable biorefining process.

Metabolismo di oligosaccaridi prebiotici in Bifidobacterium per il potenziale sviluppo di nuovi prodotti alimentari funzionali

The growth and the metabolism of Bifidobacterium adolescentis MB 239 fermenting GOS, lactose, galactose, and glucose were investigated. An unstructerd unsegregated model for growth of B. adolescentis MB 239 in batch cultures was developed and kinetic parameters were calculated with a Matlab algorithm. Galactose was the best carbon source; lactose and GOS led to lower growth rate and cellular yield, but glucose was the poorest carbon source. Lactate, acetate and ethanol yields allowed calculation of the carbon fluxes toward fermentation products. Similar distribution between 3- and 2-carbon products was observed on all the carbohydrates (45 and 55%, respectively), but ethanol production was higher on glucose than on GOS, lactose and galactose, in decreasing order. Based on the stoichiometry of the fructose 6-phosphate shunt and on the carbon distribution among the products, ATP yield was calculated on the different carbohydrates. ATP yield was the highest on galactose, while it was 5, 8, and 25% lower on lactose, GOS, and glucose, respectively. Therefore, a correspondance among ethanol production, low ATP yields, and low biomass production was established demonstrating that carbohydrate preferences may result from different sorting of carbon fluxes through the fermentative pathway. During GOS fermentation, stringent selectivity based on the degree of polymerization was exhibited, since lactose and the trisaccharide were first to be consumed, and a delay was observed until longer oligosaccharides were utilized. Throughout the growth on both lactose and GOS, galactose accumulated in the cultural broth, suggesting that β-(1-4) galactosides can be hydrolysed before they are taken up. The physiology of Bifidobacterium adolescentis MB 239 toward xylooligosaccharides (XOS) was also studied and our attention was focused on an extracellular glycosyl-hydrolase (β-Xylosidase) expressed by a culture of B. adolescentis grown on XOS as sole carbon source. The extracellular enzyme was purified from the the supernatant, which was dialyzed and concentrated by ultrafiltration. A two steps purification protocol was developed: the sample was loaded on a Mono-Q anion exchange chromatography and then, the active fractions were pooled and β-Xylosidase was purified by gel filtration chromatography on a Superdex-75. The enzyme was characterized in many aspects. β- Xylosidase was an homo-tetramer of 160 kDa as native molecular mass; it was a termostable enzyme with an optimum of temperature at 53 °C and an optimum of pH of 6.0. The kinetics parameter were calculated: km = 4.36 mM, Vmax = 0.93 mM/min. The substrate specificity with different di-, oligo- and polysaccharides was tested. The reactions were carried out overnight at pH 7 and at the optimum of temperature and the carbohydrates hydrolysis were analyzed by thin layer chromatography (TLC). Only glycosyl-hydrolase activities on XOS and on xylan were detected, whereas sucrose, lactose, cellobiose, maltose and raffinose were not hydrolyzed. It’s clearly shown that β-Xylosidase activity was higher than the Xylanase one. These studies on the carbohydrate preference of a strain of Bifidobacterium underlined the importance of the affinity between probiotics and prebiotics. On the basis of this concept, together with Barilla G&R f.lli SpA, we studied the possibility to develop a functional food containing a synbiotic. Three probiotic strains Lactobacillus plantarum BAR 10, Streptococcus thermophilus BAR 20, and Bifidobacterium lactis BAR 30 were studied to assess their suitability for utilization in synbiotic products on the basis of antioxidative activity, glutathione production, acid and bile tolerance, carbohydrates fermentation and viability in food matrices. Bile and human gastric juice resistance was tested in vitro to estimate the transit tolerance in the upper gastrointestinal tract. B. lactis and L. plantarum were more acid tolerant than S. thermophilus. All the strains resisted to bile. The growth kinetics on 13 prebiotic carbohydrates were determined. Galactooligosaccharides and fructo-oligosaccharides were successfully utilized by all the strains and could be considered the most appropriate prebiotics to be used in effective synbiotic formulations. The vitality of the three strains inoculated in different food matrices and maintained at room temperature was studied. The best survival of Lactobacillus plantarum BAR 10, Streptococcus thermophilus BAR 20, and Bifidobacterium lactis BAR 30 was found in food chocolate matrices. Then an in vivo clinical trial was carried out for 20 healthy volunteers. The increase in faecal bifidobacteria and lactobacilli populations and the efficacy of the pre-prototype was promising for the future develop of potential commercial products.