The overall objective of this PhD was to investigate the possibility to increase the nutritional value of confectionary products by the use of natural ingredients with healthy functions. The first part of the thesis focused on the possible substitution of the most characteristic component of confectionary products, i.e. refined sugar. Many natural whole sweetening alternatives are available, though not widely used; the use of molasses, the byproduct of sugar beet and cane production, still rich in healthy components as minerals and phytochemicals is hereby discussed; after having verified molasses effectiveness in oxidative stress counteraction on liver cultured cells, the higher antioxidant capacity of a sweet food prepared with molasses instead of refined sugar was confirmed. A second step of the project dealt with another main ingredient of various sweet products, namely wheat. Particularly, the exploitation of soft and durum wheat byproducts could be another sustainable strategy to improve the healthy value of confectionery. The isolation of oligosaccharides with bioactive functions form different fractions of the wheat milling stream was studied and the new ingredients were shown to have a high dietary fiber and antioxidants content. As valid alternative, product developers should consider the appealing and healthy addition of ancient grains flour to sweet baked goods. The possibility of substituting the modern whole durum wheat with the ancient Kamut® khorasan was considered, and the antioxidant and anti-inflammatory effects of these grains were evaluated and compared both in vitro and in vivo on rats. Finally, since high consumption of confectionery is a risk factor for obesity, a possible strategy for the counteraction of this disease was investigated. The ability of three bioactives in inhibiting adipocytes differentiation was investigated. In fact, theoretically, compounds able to influence adipogenesis could be used in the formulation of functional sweet products and contribute to prevent obesity.

Ischemic preconditioning is a complex cardioprotective phenomenon that involves adaptive changes in cells and molecules. This adaptation occurs in a biphasic pattern: an early phase which develops after 1-2 h, and a late phase that develops after 12-24 h. While it is widely accepted that reactive oxygen species (ROS) are strongly involved in triggering ischemic preconditiong, it is not clear if they play a major role in the early or late phase of preconditioning and which are the mechanisms involved. Methylglyoxal, a metabolic compound formed mainly from the glycolytic intermediate glyceraldehyde-3-phosphate., is a precursor of advanced glycation end product (AGEs) .It is more reactive than glucose and shows a stronger ability to cross-link with protein amino groups to form AGEs. Methylglyoxal induced cytotoxicity may be at least partially responsible for cardiovascular and Alzheimer diseases. Methylglyoxal omeostasis is controlled by the glyoxalase system that consists of two enzyme, glyoxalase 1 (GLO1) and glyoxalase 2. In a recent study it was demonstrated that the transcriptional levels of GLO1 are controlled by NF-E2-related factor 2 (Nrf2). The isothiocyanate sulforaphane, derived from the hydrolysis of glucoraphanin abundantly present in broccoli, represents one of the most potent inducers of phase II enzymes through the Keap1–Nrf2 pathway. The aim of this thesis was evaluated molecular mechanisms in cardio- and neuroprotection and the possibility of modulation by nutraceutical phytocomponents This thesis show to one side that the protection induced by H2O2 is mediated by detoxifying and antioxidant phase II enzymes induction, regulated, not only by transcriptional factor Nrf2, but also by Nrf1; on the other side our data represent an innovative result because for the first time it was demonstrated the possibility of inducing GLO1 by SF supplementation.

Previous studies in the group led to the identification of CD4+FOXP3- cells with regulatory functions in human blood that coproduce IL-10 and IFN-gamma. These cells do not belong to the Treg cell lineage since they are Foxp3- but they show some similarities with Th1 cells since they express CCR5, T-bet and produce high levels of IFN-gamma. Thus, they share relevant characteristics with both T regulatory type I cells (Tr1) and Th1 cells and we called them Th1-10 cells. In this study we presented a molecular characterization of Th1-10 cells that includes a gene expression and a microRNA profiling and performed functional studies to assess Th1-10 cells regulatory properties. We demonstrated that Th1-10 cells have a high regulatory potential being able to block the proliferation of activated CD4 naïve T cells to a similar extent as conventional Treg cells, and that this suppression capacity is at least partially mediated by secreted IL10. We showed also that Th1-10 cells are closely related to Th1 effector memory cells and express genes involved in cytotoxicity. In particular, they express the transcription factor EOMES and the cytotoxic effector molecules GZMA and GZMK, and they release cytotoxic granules upon stimulation. Moreover, we found that Eomes regulates cytotoxic functions in CD4+ T cells. We demonstrated that miR-92a, selectively downregulated in Th1-10 cells, directly targets the 3’UTR of EOMES.and this finding identifies miR-92a as a possible mediator of Th1-10 cytotoxicity. Th1-10 cells retain some proliferative capacity when sorted ex vivo and activated in vitro via their TCR, and this effect is markedly enhanced by IL-15, which also had a pro-survival effect on Th-10 cells. Thus, in contrast to conventional cytotoxic T cells, Th1-10 cells have cytotoxic and regulatory functions and are not terminally differentiated, since they retain proliferative capacity.

Evidence accumulated in the last ten years has demonstrated that a large proportion of the mitochondrial respiratory chain complexes in a variety of organisms is arranged in supramolecular assemblies called supercomplexes or respirasomes. Besides conferring a kinetic advantage (substrate channeling) and being required for the assembly and stability of Complex I, indirect considerations support the view that supercomplexes may also prevent excessive formation of reactive oxygen species (ROS) from the respiratory chain. Following this line of thought we have decided to directly investigate ROS production by Complex I under conditions in which the complex is arranged as a component of the supercomplex I1III2 or it is dissociated as an individual enzyme. The study has been addressed both in bovine heart mitochondrial membranes and in reconstituted proteoliposomes composed of complexes I and III in which the supramolecular organization of the respiratory assemblies is impaired by: (i) treatment either of bovine heart mitochondria or liposome-reconstituted supercomplex I-III with dodecyl maltoside; (ii) reconstitution of Complexes I and III at high phospholipids to protein ratio. The results of this investigation provide experimental evidence that the production of ROS is strongly increased in either model; supporting the view that disruption or prevention of the association between Complex I and Complex III by different means enhances the generation of superoxide from Complex I . This is the first demonstration that dissociation of the supercomplex I1III2 in the mitochondrial membrane is a cause of oxidative stress from Complex I. Previous work in our laboratory demonstrated that lipid peroxidation can dissociate the supramolecular assemblies; thus, here we confirm that preliminary conclusion that primary causes of oxidative stress may perpetuate reactive oxygen species (ROS) generation by a vicious circle involving supercomplex dissociation as a major determinant.

The de novo construction of cellular life requires, in part, the assembly of components that confer the ability to replicate. Herein we describe efforts to reconstitute parts of the Escherichia coli cell division machinery inside of water-in-oil emulsion compartments and synthetic phospholipid vesicles. The system was built with DNA and purified transcription and translation machinery housed in a compartment. A particular emphasis was placed on FtsZ, a protein that oligomerizes into a ring at the midcell and splits the cell into two. FtsZ does not contain a membrane interaction domain. In vivo, FtsZ interactions with the membrane are mediated by FtsA and ZipA. Therefore, the influence of FtsA on the behavior of FtsZ also was investigated. Fluorescently tagged constructs were used to facilitate evaluation by microscopy. The data showed that FtsZ readily assembles into rings in the presence of FtsA, thereby suggesting that the Fts system can be exploited for building a genetically encoded, self-replicating, cell-like system. We also explored additional methods of dividing compartments, such as the use of aqueous two and three phase systems.

The absence of clear criteria to recognize life and evaluate attempts at building a cell from component parts has slowed progress towards the construction of cellular mimics that fully display the properties of natural living cells. In the first part of this PhD thesis, a method to objectively quantify progress is proposed. In the second part of the thesis, preliminary results are shown and discussed for the construction of out-of-equilibrium cellular mimics generated by thermal gradients that do not rely on compartments made from lipid membranes.

Prion diseases are rare neurodegenerative disorders affecting humans and other animals, caused by a proteinaceous infectious agent named prion. The pivotal event in these pathologies is the conversion of PrPC, a physiologically expressed protein of poorly characterized function, into a misfolded conformer, named PrPSc, which is capable of replicating its conformationally-encoded information by inducing the conversion of its physiological counterpart. The aggregates resulting from this misfolding process accumulate in the central nervous system of affected organisms leading to neuronal death. Prion diseases are always fatal and no therapy is currently available. The lack of an effective therapeutic strategy to tackle such conditions is the result of the poor available information regarding many aspects of PrPSc, such as its structure, pathogenicity, and its replication mechanism. To complicate things further, PrPSc can appear as a set of distinct conformers, named strains, characterized by the capacity to evolve through modification and selection of their conformations, promoting resistance to treatments. In this work, we focus on two main aspects of prion biology, the elucidation of prion structure and propagation, and the development of a novel pharmacological strategy to tackle prion diseases. In both projects, we exploited the potential of integrative schemes combining computational methods and experimental data. Such approaches allowed us to build a plausible atomistic model of PrPSc and to propose a propagation mechanism describing the series of events underlying prion propagation. Moreover, the application of advanced computational schemes enabled us to identify a PrP folding intermediate displaying unique druggability properties. By exploiting the structural information of this protein conformer we identified a compound capable of acting as a pharmacological degrader for PrP by interfering with its folding pathway. Overall, this work highlights how the integration of computational and experimental methods is an extremely valuable scheme to answer complex biological questions, such as unraveling the mechanisms of protein misfolding and providing the tools to design pharmacological strategies for untreatable diseases.

Leber’s hereditary optic neuropathy (LHON) and Autosomal Dominant Optic Atrophy (ADOA) are the two most common inherited optic neuropathies and both are the result of mitochondrial dysfunctions. Despite the primary mutations causing these disorders are different, being an mtDNA mutation in subunits of complex I in LHON and defects in the nuclear gene encoding the mitochondrial protein OPA1 in ADOA, both pathologies share some peculiar features, such a variable penetrance and tissue-specificity of the pathological processes. Probably, one of the most interesting and unclear aspect of LHON is the variable penetrance. This phenomenon is common in LHON families, most of them being homoplasmic mutant. Inter-family variability of penetrance may be caused by nuclear or mitochondrial ‘secondary’ genetic determinants or other predisposing triggering factors. We identified a compensatory mechanism in LHON patients, able to distinguish affected individuals from unaffected mutation carriers. In fact, carrier individuals resulted more efficient than affected subjects in increasing the mitochondrial biogenesis to compensate for the energetic defect. Thus, the activation of the mitochondrial biogenesis may be a crucial factor in modulating penetrance, determining the fate of subjects harbouring LHON mutations. Furthermore, mtDNA content can be used as a molecular biomarker which, for the first time, clearly differentiates LHON affected from LHON carrier individuals, providing a valid mechanism that may be exploited for development of therapeutic strategies. Although the mitochondrial biogenesis gained a relevant role in LHON pathogenesis, we failed to identify a genetic modifying factor for the variable penetrance in a set of candidate genes involved in the regulation of this process. A more systematic high-throughput approach will be necessary to select the genetic variants responsible for the different efficiency in activating mitochondrial biogenesis. A genetic modifying factor was instead identified in the MnSOD gene. The SNP Ala16Val in this gene seems to modulate LHON penetrance, since the Ala allele in this position significantly predisposes to be affected. Thus, we propose that high MnSOD activity in mitochondria of LHON subjects may produce an overload of H2O2 for the antioxidant machinery, leading to release from mitochondria of this radical and promoting a severe cell damage and death ADOA is due to mutation in the OPA1 gene in the large majority of cases. The causative nuclear defects in the remaining families with DOA have not been identified yet, but a small number of families have been mapped to other chromosomal loci (OPA3, OPA4, OPA5, OPA7, OPA8). Recently, a form of DOA and premature cataract (ADOAC) has been associated to pathogenic mutations of the OPA3 gene, encoding a mitochondrial protein. In the last year OPA3 has been investigated by two different groups, but a clear function for this protein and the pathogenic mechanism leading to ADOAC are still unclear. Our study on OPA3 provides new information about the pattern of expression of the two isoforms OPA3V1 and OPA3V2, and, moreover, suggests that OPA3 may have a different function in mitochondria from OPA1, the major site for ADOA mutations. In fact, based on our results, we propose that OPA3 is not involved in the mitochondrial fusion process, but, on the contrary, it may regulate mitochondrial fission. Furthermore, at difference from OPA1, we excluded a role for OPA3 in mtDNA maintenance and we failed to identify a direct interaction between OPA3 and OPA1. Considering the results from overexpression and silencing of OPA3, we can conclude that the overexpression has more drastic consequences on the cells than silencing, suggesting that OPA3 may cause optic atrophy via a gain-of-function mechanism. These data provide a new starting point for future investigations aimed at identifying the exact function of OPA3 and the pathogenic mechanism causing ADOAC.

Cell-free expression systems are widely used to synthesize proteins for subsequent further characterization, to manufacture potentially useful commercial end products, and to construct cellular mimics in the laboratory. The first part of the thesis explores the feasibility of preparing two of the commercially available and widely used E. coli-based cell-free expression systems: the PURE System and the S30 Bacterial Extract. The second part focuses on the characterization of in vitro transcription and translation. The third part of the thesis features an example of an application of S30 Bacterial Extract cell-free expression systems i.e. the building of cell-like structures that can work together with engineered bacteria to achieve a predetermined task. Finally, the construction of a microfluidic dialysis device compatible with cell-free synthetic biology projects is presented.

Mass spectrometry imaging (MSI) is a MS-based technique. It provides a way of ascertaining both spatial distribution and relative abundance of a large variety of analytes from various biological sample surfaces. MSI is able to generate distribution maps of multiple analytes simultaneously without any labeling and does not require a prior knowledge of the target analytes, thus it has become an attractive molecular histology tool. MSI has been widely used in medicine and pharmaceutical fields, while its application in plants is recent although information regarding the spatial organization of metabolic processes in plants is of great value for understanding biological questions such as plant development, plant environment interactions, gene function and regulatory processes. The application of MSI to these studies, however, is not straightforward due to the inherent complexity of the technique. In this thesis, the issues of plant sample preparation, surface properties heterogeneity, fast MSI analysis for spatially resolved population studies and data analysis are addressed. More specifically, two MSI approaches, namely matrix assisted laser desorption ionization (MALDI) imaging and desorption electrospray ionization (DESI) imaging, have been evaluated and compared by mapping the localization of a range of secondary and primary metabolites in apple and grapes, respectively. The work based on MALDI has been focused on the optimization of sample preparation for apple tissues to preserve the true quantitative localization of metabolites and on the development of specific data analysis tool to enhance the chemical identification in untargeted MSI (chapter 3). MALDI imaging allows high-spatial localization analysis of metabolites, but it is not suitable for applications where rapid and high throughput analysis is required when the absolute quantitative information is not necessary as in the case of screening a large number of lines in genomic or plant breeding programs. DESI imaging, in contrast, is suitable for high throughput applications with the potential of obtaining statistically robust results. However, DESI is still in its infancy and there are several fundamental aspects which have to be investigated before using it as a reliable technique in extensive imaging applications. With this in mind, we investigated how DESI imaging can be used to map the distribution of the major organic acids in different grapevine tissue parts, aiming at statistically comparing their distribution differences among various grapevine tissues and gaining insights into their metabolic pathways in grapevine. Our study demonstrated that this class of molecules can be successfully detected in grapevine stem sections, but the surface property differences within the structurally heterogeneous grapevine tissues can strongly affect their semi-quantitative detection in DESI, thereby masking their true distribution. Then we decided to investigate this phenomenon in details, in a series of dedicated imaging studies, and the results have been presented in chapter 4. At the same time, during DESI experiments we have observed the production of the dianions of small dicarboxylates acids. We further studied the mechanism of formation of such species in the ion source proposing the use of doubly charged anions as a possible proxy to visualize the distributions of organic acid salts directly in plant tissues (chapter 5). The structural organization of the PhD thesis is as below: Chapter one and Chapter two describe the general MSI principle, compare the most widely used MSI ion sources, and discuss the current status in MSI data pre-processing and statistical methods. Due to the importance of sample preparation in MSI, sample handling for plant samples is independently reviewed in chapter two, with all the essential steps being fully discussed. The first two chapters describe the comprehensive picture regarding to MSI in plants. Chapter three presents high spatial and high mass resolution MALDI imaging of flavonols and dihydrochalcones in apple. Besides its importance in plant research, our results demonstrate that how data analysis as such Intensity Correlation Analysis could benefit untargeted MSI analysis. Chapter four discusses how sample surface property differences in a structurally/biologically heterogeneous sample affect the quantitative mapping of analytes in the DESI imaging of organic acids in grapevine tissue sections. Chapter five discusses the mechanism of formation of dicarboxylate dianions in DESI and ESI Chapter six summarizes the work in the thesis and discusses the future perspectives.