Ricerca, mediante tecniche di proteomica, di biomarcatori proteici utili nella valutazione della qualità degli alimenti di origine animale

In the last decades, the increase of industrial activities and of the request for the world food requirement, the intensification of natural resources exploitation, directly connected to pollution, have aroused an increasing interest of the public opinion towards initiatives linked to the regulation of food production, as well to the institution of a modern legislation for the consumer guardianship. This work was planned taking into account some important thematics related to marine environment, collecting and showing the data obtained from the studies made on different marine species of commercial interest (Chamelea gallina, Mytilus edulis, Ostrea edulis, Crassostrea gigas, Salmo salar, Gadus morhua). These studies have evaluated the effects of important physic and chemical parameters variations (temperature, xenobiotics like drugs, hydrocarbons and pesticides) on cells involved in the immune defence (haemocytes) and on some important enzymatic systems involved in xenobiotic biotransformation processes (cytochrome P450 complex) and in the related antioxidant defence processes (Superoxide dismutase, Catalase, Heat Shock Protein), from a biochemical and bimolecular point of view. Oxygen is essential in the biological answer of a living organism. Its consume in the normal cellular breathing physiological processes and foreign substances biotransformation, leads to reactive oxygen species (ROS) formation, potentially toxic and responsible of biological macromolecules damages with consequent pathologies worsening. Such processes can bring to a qualitative alteration of the derived products, but also to a general state of suffering that in the most serious cases can provoke the death of the organism, with important repercussions in economic field, in the output of the breedings, of fishing and of aquaculture. In this study it seemed interesting to apply also alternative methodologies currently in use in the medical field (cytofluorimetry) and in proteomic studies (bidimensional electrophoresis, mass spectrometry) with the aim of identify new biomarkers to place beside the traditional methods for the control of the animal origin food quality. From the results it’s possible to point out some relevant aspects from each experiment: 1. The cytofluorimetric techniques applied to O. edulis and C. gigas could bring to important developments in the search of alternative methods that quickly allows to identify with precision the origin of a specific sample, contributing to oppose possible alimentary frauds, in this case for example related to presence of a different species, also under a qualitative profile, but morpholgically similar. A concrete perspective for the application in the inspective field of this method has to be confirmed by further laboratory tests that take also in account in vivo experiments to evaluate the effect in the whole organism of the factors evaluated only on haemocytes in vitro. These elements suggest therefore the possibility to suit the cytofluorimetric methods for the study of animal organisms of food interest, still before these enter the phase of industrial working processes, giving useful information about the possible presence of contaminants sources that can induce an increase of the immune defence and an alteration of normal cellular parameter values. 2. C. gallina immune system has shown an interesting answer to benzo[a]pyrene (B[a]P) exposure, dose and time dependent, with a significant decrease of the expression and of the activity of one of the most important enzymes involved in the antioxidant defence in haemocytes and haemolymph. The data obtained are confirmed by several measurements of physiological parameters, that together with the decrease of the activity of 7-etossi-resourifine-O-deetilase (EROD linked to xenobiotic biotransformation processes) during exposure, underline the major effects of B[a]P action. The identification of basal levels of EROD supports the possible presence of CYP1A subfamily in the invertebrates, still today controversial, never identified previously in C. gallina and never isolated in the immune cells, as confirmed instead in this study with the identification of CYP1A-immunopositive protein (CYP1A-IPP). This protein could reveal a good biomarker at the base of a simple and quick method that could give clear information about specific pollutants presence, even at low concentrations in the environment where usually these organisms are fished before being commercialized. 3. In this experiment it has been evaluated the effect of the antibiotic chloramphenicol (CA) in an important species of commercial interest, Chamelea gallina. Chloramphenicol is a drug still used in some developing countries, also in veterinary field. Controls to evaluate its presence in the alimentary products of animal origin, can reveal ineffective whereas the concentration results to be below the limit of sensitivity of the instruments usually used in this type of analysis. Negative effects of CA towards the CYP1A- IPP proteins, underlined in this work, seem to be due to the attack of free radicals resultant from the action of the antibiotic. This brings to a meaningful alteration of the biotransformation mechanisms through the free radicals. It seems particularly interesting to pay attention to the narrow relationships in C. gallina, between SOD/CAT and CYP450 system, actively involved in detoxification mechanism, especially if compared with the few similar works today present about mollusc, a group that is composed by numerous species that enter in the food field and on which constant controls are necessary to evaluate in a rapid and effective way the presence of possible contaminations. 4. The investigations on fishes (Gadus morhua, and Salmo salar) and on a bivalve mollusc (Mytilus edulis) have allowed to evaluate different aspects related to the possibility to identify a biomarker for the evaluation of the health of organisms of food interest and consequently for the quality of the final product through 2DE methodologies. In the seafood field these techniques are currently used with a discreet success only for vertebrates (fishes), while in the study of the invertebrates (molluscs) there are a lot of difficulties. The results obtained in this work have underline several problems in the correct identification of the isolated proteins in animal organisms of which doesn’t currently exist a complete genomic sequence. This brings to attribute some identities on the base of the comparison with similar proteins in other animal groups, incurring in the possibility to obtain inaccurate data and above all discordant with those obtained on the same animals by other authors. Nevertheless the data obtained in this work after MALDI-ToF analysis, result however objective and the spectra collected could be again analyzed in the future after the update of genomic database related to the species studied. 4-A. The investigation about the presence of HSP70 isoforms directly induced by different phenomena of stress like B[a]P presence, has used bidimensional electrophoresis methods in C. gallina, that have allowed to isolate numerous protein on 2DE gels, allowing the collection of several spots currently in phase of analysis with MALDI-ToF-MS. The present preliminary work has allowed therefore to acquire and to improve important methodologies in the study of cellular parameters and in the proteomic field, that is not only revealed of great potentiality in the application in medical and veterinary field, but also in the field of the inspection of the foods with connections to the toxicology and the environmental pollution. Such study contributes therefore to the search of rapid and new methodologies, that can increase the inspective strategies, integrating themselves with those existing, but improving at the same time the general background of information related to the state of health of the considered animal organism, with the possibility, still hypothetical, to replace in particular cases the employment of the traditional techniques in the alimentary field.

Campionamento e determinazione di sostanze rilevanti per la valutazione della capacità ossidante dell’atmosfera e studio del loro effetto sui materiali ceramici

Carbonyl compounds are very important volatil organic compounds receiving increasing attention due to their critical role in tropospheric organic chemistry and to their effects on human health. They are first photo-oxidation stage of almost all organic compounds and originators of free radicals (OH, O3...). Therfore, continuous determination of their atmospheric concentrations is essential. Currently, field measurements are very limited mainly because of the analytical challenges posed by trace concentrations and interferences arising from atmospheric co-pollutants. Additionally, hydroxyl radical (OH) plays a central role in atmospheric chemistry processes. The measurement of atmospheric OH concentrations is essential for the comprehension of atmospheric chemical and photochemical processes. In this context, objectives of this work were: To perform a continuous survey of atmospheric concentrations of carbonyl compounds on different sites around the area of Bologna. For this purpose, an automatic instrument for the continuous measurement (one hour of time resolution) was developed and used. Additionally, the development of a complete and original analytical method was realised. To develop an original OH generation system in order to evaluate the effect of OH and the reactivity of solid and/or liquid materials of interest. This included the validation of an analytical method for the quantification of OH concentrations generated. Effect of OH on ceramic materials and reactivity of snow surface samples, involved as sink of OH in atmospheric chemistry, were evaluated.

Biological effects of bioactive components and extracts derived from edible plants commonly used in human nutrition

The main aim of this PhD research project was the evaluation of the biological effects of bioactive compounds derived from edible plants, with particular attention on their possibility to counteract oxidative damage and inflammation. After a preliminary study of in vitro antioxidant activity, regarding the modification eventually occurring after home freezing and cooking of edible vegetables, cultured mammalian cells were used as experimental model systems. Soluble extract and essential oils derived from different cultivars of Brassicaceae and Lamiaceae were tested as possible tools for the counteraction of the oxidative damage due to reactive oxygen species (ROS), underlining differences related to cultivar and agronomic techniques. Since accumulating evidence indicates that phytochemicals exhibit several additional properties in complex biological systems, a nutrigenomic approach was used to further explain the biological activity of a green tea extract, and to evidence the anti-inflammatory role of bioactive compounds derived from different foods. Overall, results obtained could contribute to a better understanding of the potential health benefit of plant foods.

Study of the components of quality in SO2-free wines obtained by innovative vinification protocols. Evaluation of the pre-fermentative addition of lysozyme and oenological tannins.

The research performed during the PhD candidature was intended to evaluate the quality of white wines, as a function of the reduction in SO2 use during the first steps of the winemaking process. In order to investigate the mechanism and intensity of interactions occurring between lysozyme and the principal macro-components of musts and wines, a series of experiments on model wine solutions were undertaken, focusing attention on the polyphenols, SO2, oenological tannins, pectines, ethanol, and sugar components. In the second part of this research program, a series of conventional sulphite added vinifications were compared to vinifications in which sulphur dioxide was replaced by lysozyme and consequently define potential winemaking protocols suitable for the production of SO2-free wines. To reach the final goal, the technological performance of two selected yeast strains with a low aptitude to produce SO2 during fermentation were also evaluated. The data obtained suggested that the addition of lysozyme and oenological tannins during the alcoholic fermentation could represent a promising alternative to the use of sulphur dioxide and a reliable starting point for the production of SO2-free wines. The different vinification protocols studied influenced the composition of the volatile profile in wines at the end of the alcoholic fermentation, especially with regards to alcohols and ethyl esters also a consequence of the yeast’s response to the presence or absence of sulphites during fermentation, contributing in different ways to the sensory profiles of wines. In fact, the aminoacids analysis showed that lysozyme can affect the consumption of nitrogen as a function of the yeast strain used in fermentation. During the bottle storage, the evolution of volatile compounds is affected by the presence of SO2 and oenological tannins, confirming their positive role in scaveging oxygen and maintaining the amounts of esters over certain levels, avoiding a decline in the wine’s quality. Even though a natural decrease was found on phenolic profiles due to oxidation effects caused by the presence of oxygen dissolved in the medium during the storage period, the presence of SO2 together with tannins contrasted the decay of phenolic content at the end of the fermentation. Tannins also showed a central role in preserving the polyphenolic profile of wines during the storage period, confirming their antioxidant property, acting as reductants. Our study focused on the fundamental chemistry relevant to the oxidative phenolic spoilage of white wines has demonstrated the suitability of glutathione to inhibit the production of yellow xanthylium cation pigments generated from flavanols and glyoxylic acid at the concentration that it typically exists in wine. The ability of glutathione to bind glyoxylic acid rather than acetaldehyde may enable glutathione to be used as a ‘switch’ for glyoxylic acid-induced polymerisation mechanisms, as opposed to the equivalent acetaldehyde polymerisation, in processes such as microoxidation. Further research is required to assess the ability of glutathione to prevent xanthylium cation production during the in-situ production of glyoxylic acid and in the presence of sulphur dioxide.

Cluster-derived Gold/Iron catalysts for environmental applications

During the last years we assisted to an exponential growth of scientific discoveries for catalysis by gold and many applications have been found for Au-based catalysts. In the literature there are several studies concerning the use of gold-based catalysts for environmental applications and good results are reported for the catalytic combustion of different volatile organic compounds (VOCs). Recently it has also been established that gold-based catalysts are potentially capable of being effectively employed in fuel cells in order to remove CO traces by preferential CO oxidation in H2-rich streams. Bi-metallic catalysts have attracted increasing attention because of their markedly different properties from either of the costituent metals, and above all their enhanced catalytic activity, selectivity and stability. In the literature there are several studies demostrating the beneficial effect due to the addition of an iron component to gold supported catalysts in terms of enhanced activity, selectivity, resistence to deactivation and prolonged lifetime of the catalyst. In this work we tried to develop a methodology for the preparation of iron stabilized gold nanoparticles with controlled size and composition, particularly in terms of obtaining an intimate contact between different phases, since it is well known that the catalytic behaviour of multi-component supported catalysts is strongly influenced by the size of the metal particles and by their reciprocal interaction. Ligand stabilized metal clusters, with nanometric dimensions, are possible precursors for the preparation of catalytically active nanoparticles with controlled dimensions and compositions. Among these, metal carbonyl clusters are quite attractive, since they can be prepared with several different sizes and compositions and, moreover, they are decomposed under very mild conditions. A novel preparation method was developed during this thesis for the preparation of iron and gold/iron supported catalysts using bi-metallic carbonyl clusters as precursors of highly dispersed nanoparticles over TiO2 and CeO2, which are widely considered two of the most suitable supports for gold nanoparticles. Au/FeOx catalysts were prepared by employing the bi-metallic carbonyl cluster salts [NEt4]4[Au4Fe4(CO)16] (Fe/Au=1) and [NEt4][AuFe4(CO)16] (Fe/Au=4), and for comparison FeOx samples were prepared by employing the homometallic [NEt4][HFe3(CO)11] cluster. These clusters were prepared by Prof. Longoni research group (Department of Physical and Inorganic Chemistry- University of Bologna). Particular attention was dedicated to the optimization of a suitable thermal treatment in order to achieve, apart from a good Au and Fe metal dispersion, also the formation of appropriate species with good catalytic properties. A deep IR study was carried out in order to understand the physical interaction between clusters and different supports and detect the occurrence of chemical reactions between them at any stage of the preparation. The characterization by BET, XRD, TEM, H2-TPR, ICP-AES and XPS was performed in order to investigate the catalysts properties, whit particular attention to the interaction between Au and Fe and its influence on the catalytic activity. This novel preparation method resulted in small gold metallic nanoparticles surrounded by highly dispersed iron oxide species, essentially in an amorphous phase, on both TiO2 and CeO2. The results presented in this thesis confirmed that FeOx species can stabilize small Au particles, since keeping costant the gold content but introducing a higher iron amount a higher metal dispersion was achieved. Partial encapsulation of gold atoms by iron species was observed since the Au/Fe surface ratio was found much lower than bulk ratio and a strong interaction between gold and oxide species, both of iron oxide and supports, was achieved. The prepared catalysts were tested in the total oxidation of VOCs, using toluene and methanol as probe molecules for aromatics and alchols, respectively, and in the PROX reaction. Different performances were observed on titania and ceria catalysts, on both toluene and methanol combustion. Toluene combustion on titania catalyst was found to be enhanced increasing iron loading while a moderate effect on FeOx-Ti activity was achieved by Au addition. In this case toluene combustion was improved due to a higher oxygen mobility depending on enhanced oxygen activation by FeOx and Au/FeOx dispersed on titania. On the contrary ceria activity was strongly decreased in the presence of FeOx, while the introduction of gold was found to moderate the detrimental effect of iron species. In fact, excellent ceria performances are due to its ability to adsorb toluene and O2. Since toluene activation is the determining factor for its oxidation, the partial coverage of ceria sites, responsible of toluene adsorption, by FeOx species finely dispersed on the surface resulted in worse efficiency in toluene combustion. Better results were obtained for both ceria and titania catalysts on methanol total oxidation. In this case, the performances achieved on differently supported catalysts indicate that the oxygen mobility is the determining factor in this reaction. The introduction of gold on both TiO2 and CeO2 catalysts, lead to a higher oxygen mobility due to the weakening of both Fe-O and Ce-O bonds and consequently to enhanced methanol combustion. The catalytic activity was found to strongly depend on oxygen mobility and followed the same trend observed for catalysts reducibility. Regarding CO PROX reaction, it was observed that Au/FeOx titania catalysts are less active than ceria ones, due to the lower reducibility of titania compared to ceria. In fact the availability of lattice oxygen involved in PROX reaction is much higher in the latter catalysts. However, the CO PROX performances observed for ceria catalysts are not really high compared to data reported in literature, probably due to the very low Au/Fe surface ratio achieved with this preparation method. CO preferential oxidation was found to strongly depend on Au particle size but also on surface oxygen reducibility, depending on the different oxide species which can be formed using different thermal treatment conditions or varying the iron loading over the support.

Biomass Gasification - Process analysis and dimensioning aspects for downdraft units and gas cleaning lines

In such territories where food production is mostly scattered in several small / medium size or even domestic farms, a lot of heterogeneous residues are produced yearly, since farmers usually carry out different activities in their properties. The amount and composition of farm residues, therefore, widely change during year, according to the single production process periodically achieved. Coupling high efficiency micro-cogeneration energy units with easy handling biomass conversion equipments, suitable to treat different materials, would provide many important advantages to the farmers and to the community as well, so that the increase in feedstock flexibility of gasification units is nowadays seen as a further paramount step towards their wide spreading in rural areas and as a real necessity for their utilization at small scale. Two main research topics were thought to be of main concern at this purpose, and they were therefore discussed in this work: the investigation of fuels properties impact on gasification process development and the technical feasibility of small scale gasification units integration with cogeneration systems. According to these two main aspects, the present work was thus divided in two main parts. The first one is focused on the biomass gasification process, that was investigated in its theoretical aspects and then analytically modelled in order to simulate thermo-chemical conversion of different biomass fuels, such as wood (park waste wood and softwood), wheat straw, sewage sludge and refuse derived fuels. The main idea is to correlate the results of reactor design procedures with the physical properties of biomasses and the corresponding working conditions of gasifiers (temperature profile, above all), in order to point out the main differences which prevent the use of the same conversion unit for different materials. At this scope, a gasification kinetic free model was initially developed in Excel sheets, considering different values of air to biomass ratio and the downdraft gasification technology as particular examined application. The differences in syngas production and working conditions (process temperatures, above all) among the considered fuels were tried to be connected to some biomass properties, such elementary composition, ash and water contents. The novelty of this analytical approach was the use of kinetic constants ratio in order to determine oxygen distribution among the different oxidation reactions (regarding volatile matter only) while equilibrium of water gas shift reaction was considered in gasification zone, by which the energy and mass balances involved in the process algorithm were linked together, as well. Moreover, the main advantage of this analytical tool is the easiness by which the input data corresponding to the particular biomass materials can be inserted into the model, so that a rapid evaluation on their own thermo-chemical conversion properties is possible to be obtained, mainly based on their chemical composition A good conformity of the model results with the other literature and experimental data was detected for almost all the considered materials (except for refuse derived fuels, because of their unfitting chemical composition with the model assumptions). Successively, a dimensioning procedure for open core downdraft gasifiers was set up, by the analysis on the fundamental thermo-physical and thermo-chemical mechanisms which are supposed to regulate the main solid conversion steps involved in the gasification process. Gasification units were schematically subdivided in four reaction zones, respectively corresponding to biomass heating, solids drying, pyrolysis and char gasification processes, and the time required for the full development of each of these steps was correlated to the kinetics rates (for pyrolysis and char gasification processes only) and to the heat and mass transfer phenomena from gas to solid phase. On the basis of this analysis and according to the kinetic free model results and biomass physical properties (particles size, above all) it was achieved that for all the considered materials char gasification step is kinetically limited and therefore temperature is the main working parameter controlling this step. Solids drying is mainly regulated by heat transfer from bulk gas to the inner layers of particles and the corresponding time especially depends on particle size. Biomass heating is almost totally achieved by the radiative heat transfer from the hot walls of reactor to the bed of material. For pyrolysis, instead, working temperature, particles size and the same nature of biomass (through its own pyrolysis heat) have all comparable weights on the process development, so that the corresponding time can be differently depending on one of these factors according to the particular fuel is gasified and the particular conditions are established inside the gasifier. The same analysis also led to the estimation of reaction zone volumes for each biomass fuel, so as a comparison among the dimensions of the differently fed gasification units was finally accomplished. Each biomass material showed a different volumes distribution, so that any dimensioned gasification unit does not seem to be suitable for more than one biomass species. Nevertheless, since reactors diameters were found out quite similar for all the examined materials, it could be envisaged to design a single units for all of them by adopting the largest diameter and by combining together the maximum heights of each reaction zone, as they were calculated for the different biomasses. A total height of gasifier as around 2400mm would be obtained in this case. Besides, by arranging air injecting nozzles at different levels along the reactor, gasification zone could be properly set up according to the particular material is in turn gasified. Finally, since gasification and pyrolysis times were found to considerably change according to even short temperature variations, it could be also envisaged to regulate air feeding rate for each gasified material (which process temperatures depend on), so as the available reactor volumes would be suitable for the complete development of solid conversion in each case, without even changing fluid dynamics behaviour of the unit as well as air/biomass ratio in noticeable measure. The second part of this work dealt with the gas cleaning systems to be adopted downstream the gasifiers in order to run high efficiency CHP units (i.e. internal engines and micro-turbines). Especially in the case multi–fuel gasifiers are assumed to be used, weightier gas cleaning lines need to be envisaged in order to reach the standard gas quality degree required to fuel cogeneration units. Indeed, as the more heterogeneous feed to the gasification unit, several contaminant species can simultaneously be present in the exit gas stream and, as a consequence, suitable gas cleaning systems have to be designed. In this work, an overall study on gas cleaning lines assessment is carried out. Differently from the other research efforts carried out in the same field, the main scope is to define general arrangements for gas cleaning lines suitable to remove several contaminants from the gas stream, independently on the feedstock material and the energy plant size The gas contaminant species taken into account in this analysis were: particulate, tars, sulphur (in H2S form), alkali metals, nitrogen (in NH3 form) and acid gases (in HCl form). For each of these species, alternative cleaning devices were designed according to three different plant sizes, respectively corresponding with 8Nm3/h, 125Nm3/h and 350Nm3/h gas flows. Their performances were examined on the basis of their optimal working conditions (efficiency, temperature and pressure drops, above all) and their own consumption of energy and materials. Successively, the designed units were combined together in different overall gas cleaning line arrangements, paths, by following some technical constraints which were mainly determined from the same performance analysis on the cleaning units and from the presumable synergic effects by contaminants on the right working of some of them (filters clogging, catalysts deactivation, etc.). One of the main issues to be stated in paths design accomplishment was the tars removal from the gas stream, preventing filters plugging and/or line pipes clogging At this scope, a catalytic tars cracking unit was envisaged as the only solution to be adopted, and, therefore, a catalytic material which is able to work at relatively low temperatures was chosen. Nevertheless, a rapid drop in tars cracking efficiency was also estimated for this same material, so that an high frequency of catalysts regeneration and a consequent relevant air consumption for this operation were calculated in all of the cases. Other difficulties had to be overcome in the abatement of alkali metals, which condense at temperatures lower than tars, but they also need to be removed in the first sections of gas cleaning line in order to avoid corrosion of materials. In this case a dry scrubber technology was envisaged, by using the same fine particles filter units and by choosing for them corrosion resistant materials, like ceramic ones. Besides these two solutions which seem to be unavoidable in gas cleaning line design, high temperature gas cleaning lines were not possible to be achieved for the two larger plant sizes, as well. Indeed, as the use of temperature control devices was precluded in the adopted design procedure, ammonia partial oxidation units (as the only considered methods for the abatement of ammonia at high temperature) were not suitable for the large scale units, because of the high increase of reactors temperature by the exothermic reactions involved in the process. In spite of these limitations, yet, overall arrangements for each considered plant size were finally designed, so that the possibility to clean the gas up to the required standard degree was technically demonstrated, even in the case several contaminants are simultaneously present in the gas stream. Moreover, all the possible paths defined for the different plant sizes were compared each others on the basis of some defined operational parameters, among which total pressure drops, total energy losses, number of units and secondary materials consumption. On the basis of this analysis, dry gas cleaning methods proved preferable to the ones including water scrubber technology in al of the cases, especially because of the high water consumption provided by water scrubber units in ammonia adsorption process. This result is yet connected to the possibility to use activated carbon units for ammonia removal and Nahcolite adsorber for chloride acid. The very high efficiency of this latter material is also remarkable. Finally, as an estimation of the overall energy loss pertaining the gas cleaning process, the total enthalpy losses estimated for the three plant sizes were compared with the respective gas streams energy contents, these latter obtained on the basis of low heating value of gas only. This overall study on gas cleaning systems is thus proposed as an analytical tool by which different gas cleaning line configurations can be evaluated, according to the particular practical application they are adopted for and the size of cogeneration unit they are connected to.

Studies of OPA1 pathogenic mechanisms in Dominant Optic Atrophy and novel protein function in mitochondrial DNA stability

The mitochondrion is an essential cytoplasmic organelle that provides most of the energy necessary for eukaryotic cell physiology. Mitochondrial structure and functions are maintained by proteins of both mitochondrial and nuclear origin. These organelles are organized in an extended network that dynamically fuses and divides. Mitochondrial morphology results from the equilibrium between fusion and fission processes, controlled by a family of “mitochondria-shaping” proteins. It is becoming clear that defects in mitochondrial dynamics can impair mitochondrial respiration, morphology and motility, leading to apoptotic cell death in vitro and more or less severe neurodegenerative disorders in vivo in humans. Mutations in OPA1, a nuclear encoded mitochondrial protein, cause autosomal Dominant Optic Atrophy (DOA), a heterogeneous blinding disease characterized by retinal ganglion cell degeneration leading to optic neuropathy (Delettre et al., 2000; Alexander et al., 2000). OPA1 is a mitochondrial dynamin-related guanosine triphosphatase (GTPase) protein involved in mitochondrial network dynamics, cytochrome c storage and apoptosis. This protein is anchored or associated on the inner mitochondrial membrane facing the intermembrane space. Eight OPA1 isoforms resulting from alternative splicing combinations of exon 4, 4b and 5b have been described (Delettre et al., 2001). These variants greatly vary among diverse organs and the presence of specific isoforms has been associated with various mitochondrial functions. The different spliced exons encode domains included in the amino-terminal region and contribute to determine OPA1 functions (Olichon et al., 2006). It has been shown that exon 4, that is conserved throughout evolution, confers functions to OPA1 involved in maintenance of the mitochondrial membrane potential and in the fusion of the network. Conversely, exon 4b and exon 5b, which are vertebrate specific, are involved in regulation of cytochrome c release from mitochondria, and activation of apoptosis, a process restricted to vertebrates (Olichon et al., 2007). While Mgm1p has been identified thanks to its role in mtDNA maintenance, it is only recently that OPA1 has been linked to mtDNA stability. Missense mutations in OPA1 cause accumulation of multiple deletions in skeletal muscle. The syndrome associated to these mutations (DOA-1 plus) is complex, consisting of a combination of dominant optic atrophy, progressive external ophtalmoplegia, peripheral neuropathy, ataxia and deafness (Amati- Bonneau et al., 2008; Hudson et al., 2008). OPA1 is the fifth gene associated with mtDNA “breakage syndrome” together with ANT1, PolG1-2 and TYMP (Spinazzola et al., 2009). In this thesis we show for the first time that specific OPA1 isoforms associated to exon 4b are important for mtDNA stability, by anchoring the nucleoids to the inner mitochondrial membrane. Our results clearly demonstrate that OPA1 isoforms including exon 4b are intimately associated to the maintenance of the mitochondrial genome, as their silencing leads to mtDNA depletion. The mechanism leading to mtDNA loss is associated with replication inhibition in cells where exon 4b containing isoforms were down-regulated. Furthermore silencing of exon 4b associated isoforms is responsible for alteration in mtDNA-nucleoids distribution in the mitochondrial network. In this study it was evidenced that OPA1 exon 4b isoform is cleaved to provide a 10kd peptide embedded in the inner membrane by a second transmembrane domain, that seems to be crucial for mitochondrial genome maintenance and does correspond to the second transmembrane domain of the yeasts orthologue encoded by MGM1 or Msp1, which is also mandatory for this process (Diot et al., 2009; Herlan et al., 2003). Furthermore in this thesis we show that the NT-OPA1-exon 4b peptide co-immuno-precipitates with mtDNA and specifically interacts with two major components of the mitochondrial nucleoids: the polymerase gamma and Tfam. Thus, from these experiments the conclusion is that NT-OPA1- exon 4b peptide contributes to the nucleoid anchoring in the inner mitochondrial membrane, a process that is required for the initiation of mtDNA replication and for the distribution of nucleoids along the network. These data provide new crucial insights in understanding the mechanism involved in maintenance of mtDNA integrity, because they clearly demonstrate that, besides genes implicated in mtDNA replications (i.e. polymerase gamma, Tfam, twinkle and genes involved in the nucleotide pool metabolism), OPA1 and mitochondrial membrane dynamics play also an important role. Noticeably, the effect on mtDNA is different depending on the specific OPA1 isoforms down-regulated, suggesting the involvement of two different combined mechanisms. Over two hundred OPA1 mutations, spread throughout the coding region of the gene, have been described to date, including substitutions, deletions or insertions. Some mutations are predicted to generate a truncated protein inducing haploinsufficiency, whereas the missense nucleotide substitutions result in aminoacidic changes which affect conserved positions of the OPA1 protein. So far, the functional consequences of OPA1 mutations in cells from DOA patients are poorly understood. Phosphorus MR spectroscopy in patients with the c.2708delTTAG deletion revealed a defect in oxidative phosphorylation in muscles (Lodi et al., 2004). An energetic impairment has been also show in fibroblasts with the severe OPA1 R445H mutation (Amati-Bonneau et al., 2005). It has been previously reported by our group that OPA1 mutations leading to haploinsufficiency are associated in fibroblasts to an oxidative phosphorylation dysfunction, mainly involving the respiratory complex I (Zanna et al., 2008). In this study we have evaluated the energetic efficiency of a panel of skin fibroblasts derived from DOA patients, five fibroblast cell lines with OPA1 mutations causing haploinsufficiency (DOA-H) and two cell lines bearing mis-sense aminoacidic substitutions (DOA-AA), and compared with control fibroblasts. Although both types of DOA fibroblasts maintained a similar ATP content when incubated in a glucose-free medium, i.e. when forced to utilize the oxidative phosphorylation only to produce ATP, the mitochondrial ATP synthesis through complex I, measured in digitonin-permeabilized cells, was significantly reduced in cells with OPA1 haploinsufficiency only, whereas it was similar to controls in cells with the missense substitutions. Furthermore, evaluation of the mitochondrial membrane potential (DYm) in the two fibroblast lines DOA-AA and in two DOA-H fibroblasts, namely those bearing the c.2819-2A>C mutation and the c.2708delTTAG microdeletion, revealed an anomalous depolarizing response to oligomycin in DOA-H cell lines only. This finding clearly supports the hypothesis that these mutations cause a significant alteration in the respiratory chain function, which can be unmasked only when the operation of the ATP synthase is prevented. Noticeably, oligomycin-induced depolarization in these cells was almost completely prevented by preincubation with cyclosporin A, a well known inhibitor of the permeability transition pore (PTP). This results is very important because it suggests for the first time that the voltage threshold for PTP opening is altered in DOA-H fibroblasts. Although this issue has not yet been addressed in the present study, several are the mechanisms that have been proposed to lead to PTP deregulation, including in particular increased reactive oxygen species production and alteration of Ca2+ homeostasis, whose role in DOA fibroblasts PTP opening is currently under investigation. Identification of the mechanisms leading to altered threshold for PTP regulation will help our understanding of the pathophysiology of DOA, but also provide a strategy for therapeutic intervention.

Cell-free cryopreserved arterial allografts from multiorgan donors: a new strategy to fabricate artificial blood vessels suited for peripheral vascular surgery

Critical lower limb ischemia is a severe disease. A common approach is infrainguinal bypass. Synthetic vascular prosthesis, are good conduits in high-flow low-resistance conditions but have difficulty in their performance as small diameter vessel grafts. A new approach is the use of native decellularized vascular tissues. Cell-free vessels are expected to have improved biocompatibility when compared to synthetic and are optimal natural 3D matrix templates for driving stem cell growth and tissue assembly in vivo. Decellularization of tissues represent a promising field for regenerative medicine, with the aim to develop a methodology to obtain small-diameter allografts to be used as a natural scaffold suited for in vivo cell growth and pseudo-tissue assembly, eliminating failure caused from immune response activation. Material and methods. Umbilical cord-derived mesenchymal cells isolated from human umbilical cord tissue were expanded in advanced DMEM. Immunofluorescence and molecular characterization revealed a stem cell profile. A non-enzymatic protocol, that associate hypotonic shock and low-concentration ionic detergent, was used to decellularize vessel segments. Cells were seeded cell-free scaffolds using a compound of fibrin and thrombin and incubated in DMEM, after 4 days of static culture they were placed for 2 weeks in a flow-bioreactor, mimicking the cardiovascular pulsatile flow. After dynamic culture, samples were processed for histological, biochemical and ultrastructural analysis. Discussion. Histology showed that the dynamic culture cells initiate to penetrate the extracellular matrix scaffold and to produce components of the ECM, as collagen fibres. Sirius Red staining showed layers of immature collagen type III and ultrastructural analysis revealed 30 nm thick collagen fibres, presumably corresponding to the immature collagen. These data confirm the ability of cord-derived cells to adhere and penetrate a natural decellularized tissue and to start to assembly into new tissue. This achievement makes natural 3D matrix templates prospectively valuable candidates for clinical bypass procedures