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.

Evaluation of glycoconjugate antigens as vaccine candidates against group A Streptococcus and human immunodeficiency virus infections

This PhD thesis discusses the rationale for design and use of synthetic oligosaccharides for the development of glycoconjugate vaccines and the role of physicochemical methods in the characterization of these vaccines. The study concerns two infectious diseases that represent a serious problem for the national healthcare programs: human immunodeficiency virus (HIV) and Group A Streptococcus (GAS) infections. Both pathogens possess distinctive carbohydrate structures that have been described as suitable targets for the vaccine design. The Group A Streptococcus cell membrane polysaccharide (GAS-PS) is an attractive vaccine antigen candidate based on its conserved, constant expression pattern and the ability to confer immunoprotection in a relevant mouse model. Analysis of the immunogenic response within at-risk populations suggests an inverse correlation between high anti-GAS-PS antibody titres and GAS infection cases. Recent studies show that a chemically synthesized core polysaccharide-based antigen may represent an antigenic structural determinant of the large polysaccharide. Based on GAS-PS structural analysis, the study evaluates the potential to exploit a synthetic design approach to GAS vaccine development and compares the efficiency of synthetic antigens with the long isolated GAS polysaccharide. Synthetic GAS-PS structural analogues were specifically designed and generated to explore the impact of antigen length and terminal residue composition. For the HIV-1 glycoantigens, the dense glycan shield on the surface of the envelope protein gp120 was chosen as a target. This shield masks conserved protein epitopes and facilitates virus spread via binding to glycan receptors on susceptible host cells. The broadly neutralizing monoclonal antibody 2G12 binds a cluster of high-mannose oligosaccharides on the gp120 subunit of HIV-1 Env protein. This oligomannose epitope has been a subject to the synthetic vaccine development. The cluster nature of the 2G12 epitope suggested that multivalent antigen presentation was important to develop a carbohydrate based vaccine candidate. I describe the development of neoglycoconjugates displaying clustered HIV-1 related oligomannose carbohydrates and their immunogenic properties.

The immunogenicity of chemically derived zwitterionic polysaccharides

Bacterial capsular polysaccharides (PS) which naturally contain zwitterionic charge motifs (ZPS) possess specific immunostimulatory activity, leading to direct activation of antigen-presenting cells (APCs) through Toll-like receptor 2 (TLR2) and of T cells in co-culture systems. When administered intraperitoneally, ZPS and bacteria expressing them are involved in the induction or regulation of T-cell dependent inflammatory processes such as intra-abdominal abscess formation. Moreover it has been published that ZPSs are processed to low molecular weight carbohydrates and presented to T cells through a pathway similar to that used for protein antigens. These findings were in contrast with the paradigm according to which polysaccharides are T-independent antigens unable to be presented in association with MHC class II molecules and unable to induce a protective immune response. For this reason in glycoconjugate vaccines polysaccharides often need to be conjugated to a carrier protein to induce protection. The aim of our work was to generate vaccine candidates with antigen and adjuvant properties in one molecule by the chemical introduction of a positive charge into naturally anionic PS from group B streptococcus (GBS). The resulting zwitterionic PS (ZPS) has the ability to activate human and mouse APCs, and in mixed co-cultures of monocytes and T cells, ZPS induce MHC II-dependent T-cell proliferation and up-regulation of activation markers. TLR2 transfectants show reporter gene transcription upon incubation with ZPS and these stimulatory qualities can be blocked by anti-TLR2 mAbs or by the destruction of the zwitterionic motif. However, in vivo, ZPS used alone as vaccine antigen failed to induce protection against GBS challenge, a result which does not confirm the above mentioned postulate that ZPS are T-cell dependent Ags by virtue of their charge motif. Thus to make ZPS visible to the immune system we have conjugated ZPS with a carrier protein. ZPS-glycoconjugates induce higher T cell and Ab responses to carrier and PS, respectively, compared to control PS-glycoconjugates made with the native polysaccharide form. Moreover, protection of mothers or neonate offspring from lethal GBS challenge is better when mothers are immunized with ZPS-conjugates compared to immunization with PS-conjugates. In TLR2 knockout mice, ZPS-conjugates lose both their increased immunogenicity and protective effect after vaccination. When ZPS are co-administered as adjuvants with unconjugated tetanus toxoid (TT), they have the ability to increase the TT-specific antibody titer. In conclusion, glycoconjugates containing ZPS are potent vaccines. They target Ag to TLR2-expressing APCs and activate these APCs, leading to better T cell priming and ultimately to higher protective Ab titers. Thus, rational chemical design can generate potent novel PS-adjuvants with wide application, including glycoconjugates and co-administration with unrelated protein Ags.

Researches on berry composition in red grape: agronomical, biochemical and molecular approaches

It is well known that the best grape quality can occur only through the achievement of optimal source/sink ratio. Vine balance is in fact a key parameter in controlling berry sugar, acidity and secondary metabolites content (Howell, 2001; Vanden Heuvel et al., 2004). Despite yield reduction and quality improvement are not always strictly related, cluster thinning is considered a technique which could lead to improvement in grape sugar and anthocyanin composition (Dokoozlian and Hirschfelt, 1995; Guidoni et al., 2002). Among several microclimatic variables which may impact grape composition, the effect of cluster light exposure and temperature, which probably act in synergistic and complex way, has been widely explored showing positive even sometimes contradictory results (Spayd et al., 2001; Tarara et al., 2008). Pre-bloom and véraison defoliation are very efficient techniques in inducing cluster microclimatic modification. Furthermore pre-bloom defoliation inducing a lower berry set percentage On these basis the aim of the first experiment of the thesis was to verify in cv Sangiovese the effects on ripening and berry composition of management techniques which may increase source/sink ratio and /or promote light incidence on berries throughout grape ripening. An integrated agronomic, biochemical and microarray approach, aims to understand which mechanisms are involved in berry composition and may be conditioned in the berries during ripening in vines submitted to three treatments. In particular the treatments compared were: a) cluster thinning (increasing in source/sink ratio) b) leaf removal at véraison (increasing cluster light exposure) c) pre-bloom defoliation (increasing source sink ratio and cluster light exposure). Vine response to leaf removal at véraison was further evaluated in the second experiment on three different varieties (Cabernet Sauvignon, Nero d’Avola, Raboso Piave) chosen for their different genetic traits in terms of anthocyanin amount and composition. The integrated agronomic, biochemical and microarray approach, employed in order to understand those mechanisms involved in berry composition of Sangiovese vines submitted to management techniques which may increase source/sink ratio and induce microclimatic changes, bring to interesting results. This research confirmed the main role of source/sink ratio in conditioning sugars metabolism and revealed also that carbohydrates availability is a crucial issue in triggering anthocyanin biosynthesis. More complex is the situation of pre-bloom defoliation, where source/sink and cluster light increase effects are associated to determine final berry composition. It results that the application of pre-bloom defoliation may be risky, as too much dependent on seasonal conditions (rain and temperature) and physiological vine response (leaf area recovery, photosynthetic compensation, laterals regrowth). Early induced stress conditions could bring cluster at véraison in disadvantage to trigger optimal berry ripening processes compared to untreated vines. This conditions could be maintained until harvest, if no previously described physiological recovery occurs. Certainly, light exposure increase linked to defoliation treatments, showed a positive and solid effect on flavonol biosynthesis, as in our conditions temperature was not so different among treatments. Except the last aspects, that could be confirmed also for véraison defoliation, microclimatic changes by themselves seemed not able to induce any modification in berry composition. Further studies are necessary to understand if the peculiar anthocyanic and flavonols composition detected in véraison defoliation could play important role in both color intensity and stability of wines.

Interaction between APOE4 genotype and environmental risk factors in Alzheimer's disease

Alzheimer's disease (AD) is probably caused by both genetic and environmental risk factors. The major genetic risk factor is the E4 variant of apolipoprotein E gene called apoE4. Several risk factors for developing AD have been identified including lifestyle, such as dietary habits. The mechanisms behind the AD pathogenesis and the onset of cognitive decline in the AD brain are presently unknown. In this study we wanted to characterize the effects of the interaction between environmental risk factors and apoE genotype on neurodegeneration processes, with particular focus on behavioural studies and neurodegenerative processes at molecular level. Towards this aim, we used 6 months-old apoE4 and apoE3 Target Replacement (TR) mice fed on different diets (high intake of cholesterol and high intake of carbohydrates). These mice were evaluated for learning and memory deficits in spatial reference (Morris Water Maze (MWM)) and contextual learning (Passive Avoidance) tasks, which involve the hippocampus and the amygdala, respectively. From these behavioural studies we found that the initial cognitive impairments manifested as a retention deficit in apoE4 mice fed on high carbohydrate diet. Thus, the genetic risk factor apoE4 genotype associated with a high carbohydrate diet seems to affect cognitive functions in young mice, corroborating the theory that the combination of genetic and environmental risk factors greatly increases the risk of developing AD and leads to an earlier onset of cognitive deficits. The cellular and molecular bases of the cognitive decline in AD are largely unknown. In order to determine the molecular changes for the onset of the early cognitive impairment observed in the behavioural studies, we performed molecular studies, with particular focus on synaptic integrity and Tau phosphorylation. The most relevant finding of our molecular studies showed a significant decrease of Brain-derived Neurotrophic Factor (BDNF) in apoE4 mice fed on high carbohydrate diet. Our results may suggest that BDNF decrease found in apoE4 HS mice could be involved in the earliest impairment in long-term reference memory observed in behavioural studies. The second aim of this thesis was to study possible involvement of leptin in AD. There is growing evidence that leptin has neuroprotective properties in the Central Nervous System (CNS). Recent evidence has shown that leptin and its receptors are widespread in the CNS and may provide neuronal survival signals. However, there are still numerous questions, regarding the molecular mechanism by which leptin acts, that remain unanswered. Thus, given to the importance of the involvement of leptin in AD, we wanted to clarify the function of leptin in the pathogenesis of AD and to investigate if apoE genotype affect leptin levels through studies in vitro, in mice and in human. Our findings suggest that apoE4 TR mice showed an increase of leptin in the brain. Leptin levels are also increased in the cerebral spinal fluid of AD patients and apoE4 carriers with AD have higher levels of leptin than apoE3 carriers. Moreover, leptin seems to be expressed by reactive glial cells in AD brains. In vitro, ApoE4 together with Amyloid beta increases leptin production by microglia and astrocytes. Taken together, all these findings suggest that leptin replacement might not be a good strategy for AD therapy. Our results show that high leptin levels were found in AD brains. These findings suggest that, as high leptin levels do not promote satiety in obese individuals, it might be possible that they do not promote neuroprotection in AD patients. Therefore, we hypothesized that AD brain could suffer from leptin resistance. Further studies will be critical to determine whether or not the central leptin resistance in SNC could affect its potential neuroprotective effects.

Identification of the N-Linked Glycosylation Sites of the Transcription Factor Rest and Effect of Glycosylation on DNA Binding and Transcriptional Activity

REST is a zinc-finger transcription factor implicated in several processes such as maintenance of embryonic stem cell pluripotency and regulation of mitotic fidelity in non-neuronal cells [Chong et al., 1995]. The gene encodes for a 116-kDa protein that acts as a molecular platform for co-repressors recruitment and promotes modifications of DNA and histones [Ballas, 2005]. REST showed different apparent molecular weights, consistent with the possible presence of post-translational modifications [Lee et al., 2000]. Among these the most common is glycosylation, the covalent attachment of carbohydrates during or after protein synthesis [Apweiler et al., 1999] My thesis has ascertained, for the first time, the presence of glycan chians in the transcription factor REST. Through enzymatic deglycosylation and MS, oligosaccharide composition of glycan chains was evaluated: a complex mixture of glycans, composed of N-acetylgalactosamine, galactose and mannose, was observed thus confirming the presence of O- and N-linked glycan chains. Glycosylation site mapping was done using a 18O-labeling method and MS/MS and twelve potential N-glycosylation sites were identified. The most probable glycosylation target residues were mutated through site-directed mutagenesis and REST mutants were expressed in different cell lines. Variations in the protein molecular weight and mutant REST ability to bind the RE-1 sequence were analyzed. Gene reporter assays showed that, altogether, removal of N-linked glycan chains causes loss of transcriptional repressor function, except for mutant N59 which showed a slight residual repressor activity in presence of IGF-I. Taken togheter these results demonstrate the presence of complex glycan chians in the transcription factor REST: I have depicted their composition, started defining their position on the protein backbone and identified their possible role in the transcription factor functioning. Considering the crucial role of glycosylation and transcription factors activity in the aetiology of many diseases, any further knowledge could find important and interesting pharmacological application.

Allevamento massale di Aedes albopictus (Skuse) nell’ambito della tecnica SIT (Tecnica dell’Insetto Sterile)

Dal 1999 presso il laboratorio del Centro Agricoltura Ambiente “G. Nicoli” a Crevalcore (BO) è in corso una sperimentazione finalizzata a verificare la possibilità di attuare la tecnica del maschio sterile (SIT) in Italia contro Aedes albopictus. Alcuni aspetti per migliorare l’efficienza di questa struttura pilota, oggetto della presente ricerca, sono stati: 1) studio degli effetti di determinati costituenti della dieta larvale a) sullo sviluppo larvale stesso, per individuare intervalli limite di densità larvale e di concentrazione di cibo in cui è possibile lo sviluppo di tale specie, e b) sulla qualità dei maschi adulti ottenuti; 2) la valutazione di attrezzatura per l’allevamento massale e 3) la possibilità di migliorare la dieta larvale mediante integrazione di carboidrati. Dalle prove di valutazione della dieta larvale si è potuto osservare che, per quanto riguarda i parametri larvali, le due diete denominate “IAEA” (1 e 2) sono risultate più efficaci rispetto alla dieta standard “CAA”. Tali diete sono perciò da preferirsi nel loro possibile impiego in biofabbriche per l’allevamento massale. Le prove condotte sugli adulti allevati con le diverse diete hanno suggerito la necessità di valutare una possibile integrazione di componenti per migliorarne la longevità. Risulta altresì opportuno continuare la ricerca per ottimizzare la dieta larvale così da ottenere maschi di elevata qualità. Grazie ai risultati ottenuti dalle prove per valutare l’impiego di attrezzatura massale (vassoi di grandi dimensioni e carrello) si è potuto definire un modello per l’allevamento di Ae. albopictus con parametri standardizzati di densità larvale, dose di dieta, temperatura dell’acqua di allevamento, percentuale di maschi passati al setacciamento e rendimento di allevamento. Prove future saranno necessarie per testare altri componenti della dieta ricchi in carboidrati, quali saccarosio, da aggiungere alla dieta larvale per migliorare le qualità degli adulti ottenuti senza provocare effetti negativi sui parametri dello sviluppo larvale.

Starch distribution in pear tree organs in relation to training systems, rootstocks and fruit quality

Starch is the main form in which plants store carbohydrates reserves, both in terms of amounts and distribution among different plant species. Carbohydrates are direct products of photosynthetic activity, and it is well know that yield efficiency and production are directly correlated to the amount of carbohydrates synthesized and how these are distributed among vegetative and reproductive organs. Nowadays, in pear trees, due to the modernization of orchards, through the introduction of new rootstocks and the development of new training systems, the understanding and the development of new approaches regarding the distribution and storage of carbohydrates, are required. The objective of this research work was to study the behavior of carbohydrate reserves, mainly starch, in different pear tree organs and tissues: i.e., fruits, leaves, woody organs, roots and flower buds, at different physiological stages during the season. Starch in fruit is accumulated at early stages, and reached a maximum concentration during the middle phase of fruit development; after that, its degradation begins with a rise in soluble carbohydrates. Moreover, relationships between fruit starch degradation and different fruit traits, soluble sugars and organic acids were established. In woody organs and roots, an interconversion between starch and soluble carbohydrates was observed during the dormancy period that confirms its main function in supporting the growth and development of new tissues during the following spring. Factors as training systems, rootstocks, types of bearing wood, and their position on the canopy, influenced the concentrations of starch and soluble carbohydrates at different sampling dates. Also, environmental conditions and cultural practices must be considered to better explain these results. Thus, a deeper understanding of the dynamics of carbohydrates reserves within the plant could provide relevant information to improve several management practices to increase crop yield efficiency.

1st and 2nd Generation Ethanol from Biomass Crops

In chapter 1 and 2 calcium hydroxide as impregnation agent before steam explosion of sugarcane bagasse and switchgrass, respectively, was compared with auto-hydrolysis, assessing the effects on enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) at high solid concentration of pretreated solid fraction. In addition, anaerobic digestion of pretreated liquid fraction was carried out, in order to appraise the effectiveness of calcium hydroxide before steam explosion in a more comprehensive way. In As water is an expensive input in both cultivation of biomass crops and subsequent pretreatment, Chapter 3 addressed the effects of variable soil moisture on biomass growth and composition of biomass sorghum. Moreover, the effect of water stress was related to the characteristics of stem juice for 1st generation ethanol and structural carbohydrates for 2nd generation ethanol. In the frame of chapter 1, calcium hydroxide was proven to be a suitable catalyst for sugarcane bagasse before steam explosion, in order to enhance fibre deconstruction. In chapter 2, effect of calcium hydroxide on switchgrass showed a great potential when ethanol was focused, whereas acid addition produced higher methane yield. Regarding chapter 3, during crop cycle the amount of cellulose, hemicellulose and AIL changed causing a decrease of 2G ethanol amount. Biomass physical and chemical properties involved a lower glucose yield and concentration at the end of enzymatic hydrolysis and, consequently, a lower 2G ethanol concentration at the end of simultaneous saccharification and fermentation, proving that there is strong relationship between structure, chemical composition, and fermentable sugar yield. The significantly higher concentration of ethanol at the early crop stage could be an important incentive to consider biomass sorghum as second crop in the season, to be introduced into some agricultural systems, potentially benefiting farmers and, above all, avoiding the exacerbation of the debate about fuel vs food crops.