Novel two photon absorbers: evaluation of photophysical properties in view of biomedical applications

This thesis was focused on the investigation of the linear optical properties of novel two photon absorbers for biomedical applications. Substituted imidazole and imidazopyridine derivatives, and organic dendrimers were studied as potential fluorophores for two photon bioimaging. The results obtained showed superior luminescence properties for sulphonamido imidazole derivatives compared to other substituted imidazoles. Imidazo[1,2-a]pyridines exhibited an important dependence on the substitution pattern of their luminescence properties. Substitution at imidazole ring led to a higher fluorescence yield than the substitution at the pyridine one. Bis-imidazo[1,2-a]pyridines of Donor-Acceptor-Donor type were examined. Bis-imidazo[1,2-a]pyridines dimerized at C3 position had better luminescence properties than those dimerized at C5, displaying high emission yields and important 2PA cross sections. Phosphazene-based dendrimers with fluorene branches and cationic charges on the periphery were also examined. Due to aggregation phenomena in polar solvents, the dendrimers registered a significant loss of luminescence with respect to fluorene chromophore model. An improved design of more rigid chromophores yields enhanced luminescence properties which, connected to large 2PA cross-sections, make this compounds valuable as fluorophores in bioimaging. The photophysical study of several ketocoumarine initiators, designed for the fabrication of small dimension prostheses by two photon polymerization (2PP) was carried out. The compounds showed low emission yields, indicative of a high population of the triplet excited state, which is the active state in producing the reactive species. Their efficiency in 2PP was proved by fabrication of microstructures and their biocompatibility was tested in the collaborator’s laboratory. In the frame of the 2PA photorelease of drugs, three fluorene-based dyads have been investigated. They were designed to release the gamma-aminobutyric acid via two photon induced electron transfer. The experimental data in polar solvents showed a fast electron transfer followed by an almost equally fast back electron transfer process, which indicate a poor optimization of the system.

Monte Carlo Simulations of Novel Scintillator Detectors and Dosimetry Calculations

Monte Carlo (MC) simulation techniques are becoming very common in the Medical Physicists community. MC can be used for modeling Single Photon Emission Computed Tomography (SPECT) and for dosimetry calculations. 188Re, is a promising candidate for radiotherapeutic production and understanding the mechanisms of the radioresponse of tumor cells "in vitro" is of crucial importance as a first step before "in vivo" studies. The dosimetry of 188Re, used to target different lines of cancer cells, has been evaluated by the MC code GEANT4. The simulations estimate the average energy deposition/per event in the biological samples. The development of prototypes for medical imaging, based on LaBr3:Ce scintillation crystals coupled with a position sensitive photomultiplier, have been studied using GEANT4 simulations. Having tested, in the simulation, surface treatments different from the one applied to the crystal used in our experimental measurements, we found out that the Energy Resolution (ER) and the Spatial Resolution (SR) could be improved, in principle, by machining in a different way the lateral surfaces of the crystal. We have then studied a system able to acquire both echographic and scintigraphic images to let the medical operator obtain the complete anatomic and functional information for tumor diagnosis. The scintigraphic part of the detector is simulated by GEANT4 and first attempts to reconstruct tomographic images have been made using as method of reconstruction a back-projection standard algorithm. The proposed camera is based on slant collimators and LaBr3:Ce crystals. Within the Field of View (FOV) of the camera, it possible to distinguish point sources located in air at a distance of about 2 cm from each other. In particular conditions of uptake, tumor depth and dimension, the preliminary results show that the Signal to Noise Ratio (SNR) values obtained are higher than the standard detection limit.

Development of screening assays to test novel integrin antagonists in allergic inflammation

Aim of the research: to develop a prototype of homogeneous high-throughput screening (HTS) for identification of novel integrin antagonists for the treatment of ocular allergy and to better understand the mechanisms of action of integrin-mediated levocabastine antiallergic action. Results: This thesis provides evidence that adopting scintillation proximity assay (SPA) levocabastine (IC50=406 mM), but not the first-generation antihistamine chlorpheniramine, displaces [125I]fibronectin (FN) binding to human a4b1 integrin. This result is supported by flow cytometry analysis, where levocabastine antagonizes the binding of a primary antibody to integrin a4 expressed in Jurkat E6.1 cells. Levocabastine, but not chlorpheniramine, binds to a4b1 integrin and prevents eosinophil adhesion to VCAM-1, FN or human umbilical vein endothelial cells (HUVEC) cultured in vitro. Similarly, levocabastine affects aLb2/ICAM-1-mediated adhesion of Jurkat E6.1 cells. Analyzing the supernatant of TNF-a-treated (24h) eosinophilic cells (EoL-1), we report that levocabastine reduces the TNF-a-induced release of the cytokines IL-12p40, IL-8 and VEGF. Finally, in a model of allergic conjunctivitis, levocastine eye drops (0.05%) reduced the clinical aspects of the early and late phase reactions and the conjunctival expression of a4b1 integrin by reducing infiltrated eosinophils. Conclusions: SPA is a highly efficient, amenable to automation and robust binding assay to screen novel integrin antagonists in a HTS setting. We propose that blockade of integrinmediated cell adhesion might be a target of the anti-allergic action of levocabastine and may play a role in preventing eosinophil adhesion and infiltration in allergic conjunctivitis.

Design and synthesis of novel non peptidomimtic beta-secretase inhibitors in the treatment of Alzheimer's disease

The aspartic protease BACE1 (β-amyloid precursor protein cleaving enzyme, β-secretase) is recognized as one of the most promising targets in the treatment of Alzheimer's disease (AD). The accumulation of β-amyloid peptide (Aβ) in the brain is a major factor in the pathogenesis of AD. Aβ is formed by initial cleavage of β-amyloid precursor protein (APP) by β-secretase, therefore BACE1 inhibition represents one of the therapeutic approaches to control progression of AD, by preventing the abnormal generation of Aβ. For this reason, in the last decade, many research efforts have focused at the identification of new BACE1 inhibitors as drug candidates. Generally, BACE1 inhibitors are grouped into two families: substrate-based inhibitors, designed as peptidomimetic inhibitors, and non-peptidomimetic ones. The research on non-peptidomimetic small molecules BACE1 inhibitors remains the most interesting approach, since these compounds hold an improved bioavailability after systemic administration, due to a good blood-brain barrier permeability in comparison to peptidomimetic inhibitors. Very recently, our research group discovered a new promising lead compound for the treatment of AD, named lipocrine, a hybrid derivative between lipoic acid and the AChE inhibitor (AChEI) tacrine, characterized by a tetrahydroacridinic moiety. Lipocrine is one of the first compounds able to inhibit the catalytic activity of AChE and AChE-induced amyloid-β aggregation and to protect against reactive oxygen species. Due to this interesting profile, lipocrine was also evaluated for BACE1 inhibitory activity, resulting in a potent lead compound for BACE1 inhibition. Starting from this interesting profile, a series of tetrahydroacridine analogues were synthesised varying the chain length between the two fragments. Moreover, following the approach of combining in a single molecule two different pharmacophores, we designed and synthesised different compounds bearing the moieties of known AChEIs (rivastigmine and caproctamine) coupled with lipoic acid, since it was shown that dithiolane group is an important structural feature of lipocrine for the optimal inhibition of BACE1. All the tetrahydroacridines, rivastigmine and caproctamine-based compounds, were evaluated for BACE1 inhibitory activity in a FRET (fluorescence resonance energy transfer) enzymatic assay (test A). With the aim to enhancing the biological activity of the lead compound, we applied the molecular simplification approach to design and synthesize novel heterocyclic compounds related to lipocrine, in which the tetrahydroacridine moiety was replaced by 4-amino-quinoline or 4-amino-quinazoline rings. All the synthesized compounds were also evaluated in a modified FRET enzymatic assay (test B), changing the fluorescent substrate for enzymatic BACE1 cleavage. This test method guided deep structure-activity relationships for BACE1 inhibition on the most promising quinazoline-based derivatives. By varying the substituent on the 2-position of the quinazoline ring and by replacing the lipoic acid residue in lateral chain with different moieties (i.e. trans-ferulic acid, a known antioxidant molecule), a series of quinazoline derivatives were obtained. In order to confirm inhibitory activity of the most active compounds, they were evaluated with a third FRET assay (test C) which, surprisingly, did not confirm the previous good activity profiles. An evaluation study of kinetic parameters of the three assays revealed that method C is endowed with the best specificity and enzymatic efficiency. Biological evaluation of the modified 2,4-diamino-quinazoline derivatives measured through the method C, allow to obtain a new lead compound bearing the trans-ferulic acid residue coupled to 2,4-diamino-quinazoline core endowed with a good BACE1 inhibitory activity (IC50 = 0.8 mM). We reported on the variability of the results in the three different FRET assays that are known to have some disadvantages in term of interference rates that are strongly dependent on compound properties. The observed results variability could be also ascribed to different enzyme origin, varied substrate and different fluorescent groups. The inhibitors should be tested on a parallel screening in order to have a more reliable data prior to be tested into cellular assay. With this aim, preliminary cellular BACE1 inhibition assay carried out on lipocrine confirmed a good cellular activity profile (EC50 = 3.7 mM) strengthening the idea to find a small molecule non-peptidomimetic compound as BACE1 inhibitor. In conclusion, the present study allowed to identify a new lead compound endowed with BACE1 inhibitory activity in submicromolar range. Further lead optimization to the obtained derivative is needed in order to obtain a more potent and a selective BACE1 inhibitor based on 2,4-diamino-quinazoline scaffold. A side project related to the synthesis of novel enzymatic inhibitors of BACE1 in order to explore the pseudopeptidic transition-state isosteres chemistry was carried out during research stage at Università de Montrèal (Canada) in Hanessian's group. The aim of this work has been the synthesis of the δ-aminocyclohexane carboxylic acid motif with stereochemically defined substitution to incorporating such a constrained core in potential BACE1 inhibitors. This fragment, endowed with reduced peptidic character, is not known in the context of peptidomimetic design. In particular, we envisioned an alternative route based on an organocatalytic asymmetric conjugate addition of nitroalkanes to cyclohexenone in presence of D-proline and trans-2,5-dimethylpiperazine. The enantioenriched obtained 3-(α-nitroalkyl)-cyclohexanones were further functionalized to give the corresponding δ-nitroalkyl cyclohexane carboxylic acids. These intermediates were elaborated to the target structures 3-(α-aminoalkyl)-1-cyclohexane carboxylic acids in a new readily accessible way.

Computational investigation of the Plasmodium falciparum fatty acid biosynthetic pathway toward the discovery of novel antimalarials

The structural peculiarities of a protein are related to its biological function. In the fatty acid elongation cycle, one small carrier protein shuttles and delivers the acyl intermediates from one enzyme to the other. The carrier has to recognize several enzymatic counterparts, specifically interact with each of them, and finally transiently deliver the carried substrate to the active site. Carry out such a complex game requires the players to be flexible and efficiently adapt their structure to the interacting protein or substrate. In a drug discovery effort, the structure-function relationships of a target system should be taken into account to optimistically interfere with its biological function. In this doctoral work, the essential role of structural plasticity in key steps of fatty acid biosynthesis in Plasmodium falciparum is investigated by means of molecular simulations. The key steps considered include the delivery of acyl substrates and the structural rearrangements of catalytic pockets upon ligand binding. The ground-level bases for carrier/enzyme recognition and interaction are also put forward. The structural features of the target have driven the selection of proper drug discovery tools, which captured the dynamics of biological processes and could allow the rational design of novel inhibitors. The model may be perspectively used for the identification of novel pathway-based antimalarial compounds.

B-type cytochromes of the DOMON domain superfamily (Novel redox elements of plant plasma membrane)

The aim of the present study is understanding the properties of a new group of redox proteins having in common a DOMON-type domain with characteristics of cytochromes b. The superfamily of proteins containing a DOMON of this type includes a few protein families. With the aim of better characterizing this new protein family, the present work addresses both a CyDOM protein (a cytochrome b561) and a protein only comprised of DOMON(AIR12), both of plant origin. Apoplastic ascorbate can be regenerated from monodehydroascorbate by a trans-plasma membrane redox system which uses cytosolic ascorbate as a reductant and comprises a high potential cytochrome b. We identified the major plasma membrane (PM) ascorbate-reducible b-type cytochrome of bean (Phaseolus vulgaris) and soybean (Glycine max) hypocotyls as orthologs of Arabidopsis auxin-responsive gene air12. The protein, which is glycosylated and glycosylphosphatidylinositol-anchored to the external side of the PM in vivo, was expressed in Pichia pastoris in a recombinant form, lacking the glycosylphosphatidylinositol-modification signal, and purified from the culture medium. Recombinant AIR12 is a soluble protein predicted to fold into a β-sandwich domain and belonging to the DOMON superfamily. It is shown to be a b-type cytochrome with a symmetrical α-band at 561 nm, to be fully reduced by ascorbate and fully oxidized by monodehydroascorbate. Redox potentiometry suggests that AIR12 binds two high-potential hemes (Em,7 +135 and +236 mV). Phylogenetic analyses reveal that the auxin-responsive genes AIR12 constitute a new family of plasma membrane b-type cytochromes specific to flowering plants. Although AIR12 is one of the few redox proteins of the PM characterized to date, the role of AIR12 in trans-PM electron transfer would imply interaction with other partners which are still to be identified. Another part of the present project was aimed at understanding of a soybean protein comprised of a DOMON fused with a well-defined b561 cytochrome domain (CyDOM). Various bioinformatic approaches show this protein to be composed of an N-terminal DOMON followed by b561 domain. The latter contains five transmembrane helices featuring highly conserved histidines, which might bind haem groups. The CyDOM has been cloned and expressed in the yeast Pichia pastoris, and spectroscopic analyses have been accomplished on solubilized yeast membranes. CyDOM clearly reveal the properties of b-type cytochrome. The results highlight the fact that CyDOM is clearly able to lead an electron flux through the plasmamembrane. Voltage clamp experiments demonstrate that Xenopus laevis oocytes transformed with CyDOM of soybean exhibit negative electrical currents in presence of an external electron acceptor. Analogous investigations were carried out with SDR2, a CyDOM of Drosophila melanogaster which shows an electron transport capacity even higher than plant CyDOM. As quoted above, these data reinforce those obtained in plant CyDOM on the one hand, and on the other hand allow to attribute to SDR2-like proteins the properties assigned to CyDOM. Was expressed in Regenerated tobacco roots, transiently transformed with infected a with chimeral construct GFP: CyDOM (by A. rhizogenes infection) reveals a plasmamembrane localization of CyDOM both in epidermal cells of the elongation zone of roots and in root hairs. In conclusion. Although the data presented here await to be expanded and in part clarified, it is safe to say they open a new perspective about the role of this group of proteins. The biological relevance of the functional and physiological implications of DOMON redox domains seems noteworthy, and it can but increase with future advances in research. Beyond the very finding, however interesting in itself, of DOMON domains as extracellular cytochromes, the present study testifies to the fact that cytochrome proteins containing DOMON domains of the type of “CyDOM” can transfer electrons through membranes and may represent the most important redox component of the plasmamembrane as yet discovered.

ATG based combination therapy - a novel clinically relevant approach to promote regulation and induce long-term allograft survival

Regulatory T cells (Treg) actively regulate alloimmune responses and promote transplantation tolerance. Polyclonal anti-thymocyte globulin (ATG), a widely used induction therapy in clinical organ transplantation, depletes peripheral T cells. However, resistance to tolerance induction is seen with certain T cell depleting strategies and is attributed to alterations in the balance of naïve, memory and regulatory T cells. Here we report a novel reagent, murine ATG (mATG), depletes T cells but preferentially spares CD25+ natural Tregs which limit skewing of T cell repertoire toward T-effector-memory (Tem) phenotype among the recovering T cells. T-cell depletion with mATG combined with CTLA4Ig and Sirolimus synergize to prolong graft survival by tipping the Treg/Tem balance further in favor of Tregs by preserving Tregs, facilitating generation of new Tregs by a conversion mechanism and limiting Tem expansion in response to alloantigen and homeostatic proliferation. These results provide the rationale for translating such novel combination therapies to promote tolerance in primate and human organ transplantation.

A novel phase variation mechanism in the meningococcus driven by a ligand-responsive repressor and differential spacing of distal promoter elements

Phase variable expression, mediated by high frequency reversible changes in the length of simple sequence repeats, facilitates adaptation of bacterial populations to changing environments and is frequently important in bacterial virulence. Here we elucidate a novel phase variable mechanism for NadA expression, an adhesin and invasin of Neisseria meningitidis. The NadR repressor protein binds to operators flanking the phase variable tract of the nadA promoter gene and contributes to the differential expression levels of phase variant promoters with different numbers of repeats, likely due to different spacing between operators. It is shown that IHF binds between these operators, and may permit looping of the promoter, allowing interaction of NadR at operators located distally or overlapping the promoter. The 4-hydroxyphenylacetic acid, a metabolite of aromatic amino acid catabolism that is secreted in saliva, induces nadA expression by inhibiting the DNA binding activity of the NadR repressor. When induced, only minor differences are evident between NadR-independent transcription levels of promoter phase variants, which are likely due to differential RNA polymerase contacts leading to altered promoter activity. These results suggest that NadA expression is under both stochastic and tight environmental-sensing regulatory control, and both regulations are mediated by the NadR repressor that and may be induced during colonization of the oropharynx where it plays a major role in the successful adhesion and invasion of the mucosa. Hence, simple sequence repeats in promoter regions may be a strategy used by host-adapted bacterial pathogens to randomly switch between expression states that may nonetheless still be induced by appropriate niche-specific signals.

Functional Genomics and Cell Biology of the Dolphin (Tursiops runcatus): Establishment of Novel Molecular Tools to Study Marine Mammals in Changing Environments

The dolphin (Tursiops truncatus) is a mammal that is adapted to life in a totally aquatic environment. Despite the popularity and even iconic status of the dolphin, our knowledge of its physiology, its unique adaptations and the effects on it of environmental stressors are limited. One approach to improve this limited understanding is the implementation of established cellular and molecular methods to provide sensitive and insightful information for dolphin biology. We initiated our studies with the analysis of wild dolphin peripheral blood leukocytes, which have the potential to be informative of the animal’s global immune status. Transcriptomic profiles from almost 200 individual samples were analyzed using a newly developed species-specific microarray to assess its value as a prognostic and diagnostic tool. Functional genomics analyses were informative of stress-induced gene expression profiles and also of geographical location specific transcriptomic signatures, determined by the interaction of genetic, disease and environmental factors. We have developed quantitative metrics to unambiguously characterize the phenotypic properties of dolphin cells in culture. These quantitative metrics can provide identifiable characteristics and baseline data which will enable identification of changes in the cells due to time in culture. We have also developed a novel protocol to isolate primary cultures from cryopreserved tissue of stranded marine mammals, establishing a tissue (and cell) biorepository, a new approach that can provide a solution to the limited availability of samples. The work presented represents the development and application of tools for the study of the biology, health and physiology of the dolphin, and establishes their relevance for future studies of the impact on the dolphin of environmental infection and stress.

Study of two novel streptococcus pneumoniae operons: pilus islet 2 and the lantibiotic operon

Analysis of publicly available genomes of Streptococcus pneumoniae has led to the identification of a new genomic element resembling gram-positive pilus islets (PIs). Here, we demonstrate that this genomic region, herein referred to as PI-2 (containing the genes pitA, sipA, pitB, srtG1, and srtG2) codes for a novel functional pilus in pneumococcus. Therefore, there are two pilus islets identified so far in this pathogen (PI-1 and PI-2). Polymerization of the PI-2 pilus requires the backbone protein PitB as well as the sortase SrtG1 and the signal peptidase-like protein SipA. PI-2 is associated with serotypes 1, 2, 7F, 19A, and 19F, considered to be emerging in both industrialized and developing countries. Interestingly, strains belonging to clonal complex 271 (CC271) contain both PI-1 and PI-2, as revealed by genome analyses. In these strains both pili are surface exposed and independently assembled. Furthermore, in vitro experiments provide evidence that the pilus encoded by PI-2 of S. pneumoniae is involved in adherence. Thus, pneumococci encode at least two types of pili that may play a role in the initial host cell contact to the respiratory tract. In addition, the pilus proteins are potential antigens for inclusion in a new generation of pneumococcal vaccines. Adherence by pili could represent important factor in bacterial community formation, since it has been demonstrated that bacterial community formation plays an important role in pneumococcal otitis media. In vitro quantification of bacterial community formation by S. pneumoniae was performed in order to investigate the possible role of pneumococcal pili to form communities. By using different growth media we were not able to see clear association between pili and community formation. But our findings revealed that strains belonging to MLST clonal complex CC15 efficiently form bacterial communities in vitro in a glucose dependent manner. We compared the genome of forty-four pneumococcal isolates discovering four open reading frames specifically associated with CC15. These four genes are annotated as members of an operon responsible for the biosynthesis of a putative lanctibiotic peptide, described to be involved in bacterial community formation. Our experiments show that the lanctibiotic operon deletion affects glucose mediated community formation in CC 15 strain INV200. Moreover, since glucose consumption during bacterial growth produce an acidic environment, we tested bacterial community formation at different pH and we showed that the lanctibiotic operon deletion affected pH mediated community formation in CC 15 strain INV200. In conclusion, these data demonstrate that the putative lanctibiotic operon is associated with pneumococcal CC 15 strains in vitro bacterial community formation.

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.

Toward a peripheral view of manufacturing networks

This doctoral thesis unfolds into a collection of three distinct articles that share an interest in supply firms, or “peripheral firms”. The three studies offer a novel theoretical perspective that I call the peripheral view of manufacturing networks. Building on the relational view literature, this new perspective identifies the supplier-based theoretical standpoint to analyze and explain the antecedents of relational rents in manufacturing networks. The first article, the namesake of the dissertation, is a theoretical contribution that explains the foundations of the “peripheral view of manufacturing networks”. The second article “Framing The Strategic Peripheries: A Novel Typology of Suppliers” is an empirical study with the aim to offer an interpretation of peripheries’ characteristics and dynamics. The third article, “What is Behind Absorptive Capacity? Dispelling the Opacity of R&D” presents an example of general theory development by using data from peripheral firms.

A novel map-matching procedure for low-sampling GPS data with applications to traffic flow analysis

An extensive sample (2%) of private vehicles in Italy are equipped with a GPS device that periodically measures their position and dynamical state for insurance purposes. Having access to this type of data allows to develop theoretical and practical applications of great interest: the real-time reconstruction of traffic state in a certain region, the development of accurate models of vehicle dynamics, the study of the cognitive dynamics of drivers. In order for these applications to be possible, we first need to develop the ability to reconstruct the paths taken by vehicles on the road network from the raw GPS data. In fact, these data are affected by positioning errors and they are often very distanced from each other (~2 Km). For these reasons, the task of path identification is not straightforward. This thesis describes the approach we followed to reliably identify vehicle paths from this kind of low-sampling data. The problem of matching data with roads is solved with a bayesian approach of maximum likelihood. While the identification of the path taken between two consecutive GPS measures is performed with a specifically developed optimal routing algorithm, based on A* algorithm. The procedure was applied on an off-line urban data sample and proved to be robust and accurate. Future developments will extend the procedure to real-time execution and nation-wide coverage.