Mitochondrial dysfunction in hereditary optic neuropathies

MITOCHONDRIAL DYSFUNCTION IN HEREDITARY OPTIC NEUROPATHIES Mitochondrial pathologies are a heterogeneous group of clinical manifestations characterized by oxidative phosphorylation impairment. At the beginning of their recognition mitochondrial pathologies were regarded as rare disorders but indeed they are more frequent than originally thought. Due to the unique mitochondria peculiarities mitochondrial pathologies can be caused by mutations in both mitochondrial and nuclear genomes. The poor knowledge of pathologic mechanism of these disorders has not allowed a real development of the “mitochondrial medicine”, that is currently limited to symptoms mitigation. Leber hereditary optic neuropathy (LHON) was the first pathology to be linked to a point mutation in the mtDNA. The mechanism by which point mutations in mitochondrial gene encoding Complex I subunits leads to optic nerve degeneration is still unknown, although is well accepted that other genetic or environmental factors are involved in the modulation of pathology, where a pivotal role is certainly played by oxidative stress. We studied the relationship between the Ala16Val dimorphism in the mitochondrial targeting sequence of nuclear gene SOD2 and the 3460/ND1 LHON mutation. Our results show that, in control population, the heterozygous SOD2 genotype is associated to a higher activity and quantity of MnSOD, particularly with respect to Val homozygotes. Furthermore, we demonstrated that LHON patients harboring at least one Ala allele are characterized by an increased MnSOD activity with respect to relative control population. Since the ATP synthesis rate – severely reduced in LHON patients lymphocytes - is not affected by the SOD2 genotype, we concluded that SOD2 gene could modulate the pathogenicity of LHON mutations through a mechanism associated to an increase of reactive oxygen species production. Autosomal dominant optic atrophy (ADOA) is a pathology linked to mutations in nuclear gene encoding Opa1, a dynamin-related protein localized in the mitochondrial matrix. Although the clinical course is slightly different, the endpoint of ADOA is exactly the same of LHON: optic nerve degeneration with specific involvement of retinal ganglion cells. Opa1 is a relatively new protein, whose major role is the regulation of mitochondrial fusion. Mitochondrial morphology is the results of the equilibrium between two opposite force: fusion and fission, two processes that have to be finely regulated in order to preserve mitochondrial and cellular physiology. We studied fibroblasts deriving from ADOA patients characterized by a new deletion in the GTPase domain of the OPA1 gene. The biochemical characterization of ADOA and control fibroblasts has concerned the evaluation of ATP synthesis rate, mitochondrial membrane potential in different metabolic conditions and the morphological status of mitochondria. Regarding ATP synthesis rate we did not find significant differences between ADOA and control fibroblasts even though a trend toward increased reduction in ADOA samples is observed when fibroblasts are grown in absence of glucose or in the medium containing gramicidin. Furthermore, we found that also in ADOA fibroblasts membrane potential is actively maintained by proton pumping of fully functional respiratory chain complexes. Our results indicate that the mutation found in the pedigree analyzed acts primary impairing the mitochondrial fusion without affecting the energy production, supporting the notion that cell function is tightly linked to mitochondrial morphology. Mitochondrial dysfunctions are acquiring great attention because of their recognized relevance not only in aging but also in age-related pathologies including cancer, cardiovascular disease, type II diabetes, and neurodegenerative disorders. The involvement of mitochondria in such detrimental pathologies that, currently, have become so common enhances the necessity of standardization of therapeutic strategies capable of rescuing the normal mitochondrial function. In order to propose an alternative treatment for energy deficiency-disorders we tested the effect of substrates capable to stimulate the substrate-level phosphorylation on viability and energy availability in different experimental models grown under different metabolic conditions. In fibroblasts, the energy defect was achieved by culturing cells in presence of oligomycin, an inhibitor of ATP synthase complex. NARP cybrids have been used as model of mitochondrial pathology. Cell viability and ATP content have been considered as parameters to assay the capability of exogenous substrate to rescue energy failure. Our results suggest that patients suffering for some forms of ATP synthase deficiency, or characterized by a deficiency in energy production, might benefit from dietary or pharmacological treatment based on supplementation of α-ketoglutarate and aspartate.

Interazioni tra Glicoproteine che mediano la penetrazione del Virus Herpes Simplex nella cellula e disegno razionale di Peptidi Inibitori

Herpes simplex virus entry into cells requires a multipartite fusion apparatus made of gD, gB and heterodimer gH/gL. gD serves as receptor-binding glycoprotein and trigger of fusion; its ectodomain is organized in a N-terminal domain carrying the receptor-binding sites, and a C-terminal domain carrying the profusion domain, required for fusion but not receptor-binding. gB and gH/gL execute fusion. To understand how the four glycoproteins cross-talk to each other we searched for biochemical defined complexes in infected and transfected cells, and in virions. We report that gD formed complexes with gB in absence of gH/gL, and with gH/gL in absence of gB. Complexes with similar composition were formed in infected and transfected cells. They were also present in virions prior to entry, and did not increase at virus fusion with cell. A panel of gD mutants enabled the preliminary location of part of the binding site in gD to gB to the aa 240-260 portion and downstream, with T306P307 as critical residues, and of the binding site to gH/gL at aa 260-310 portion, with P291P292 as critical residues. The results indicate that gD carries composite independent binding sites for gB and gH/gL, both of which partly located in the profusion domain. The second part of the project dealt with rational design of peptides inhibiting virus entry has been performed. Considering gB and gD, the crystal structure is known, so we designed peptides that dock in the structure or prevent the adoption of the final conformation of target molecule. Considering the other glycoproteins, of which the structure is not known, peptide libraries were analyzed. Among several peptides, some were identified as active, designed on glycoprotein B. Two of them were further analyzed. We identified peptide residues fundamental for the inhibiting activity, suggesting a possible mechanism of action. Furthermore, changing the flexibility of peptides, an increased activity was observed,with an EC50 under 10μM. New approaches will try to demonstrate the direct interaction between these peptides and the target glycoprotein B.

Development of human visual system analysis for the implementation of a new instrument for flicker measurement

Flicker is a power quality phenomenon that applies to cycle instability of light intensity resulting from supply voltage fluctuation, which, in turn can be caused by disturbances introduced during power generation, transmission or distribution. The standard EN 61000-4-15 which has been recently adopted also by the IEEE as IEEE Standard 1453 relies on the analysis of the supply voltage which is processed according to a suitable model of the lamp – human eye – brain chain. As for the lamp, an incandescent 60 W, 230 V, 50 Hz source is assumed. As far as the human eye – brain model is concerned, it is represented by the so-called flicker curve. Such a curve was determined several years ago by statistically analyzing the results of tests where people were subjected to flicker with different combinations of magnitude and frequency. The limitations of this standard approach to flicker evaluation are essentially two. First, the provided index of annoyance Pst can be related to an actual tiredness of the human visual system only if such an incandescent lamp is used. Moreover, the implemented response to flicker is “subjective” given that it relies on the people answers about their feelings. In the last 15 years, many scientific contributions have tackled these issues by investigating the possibility to develop a novel model of the eye-brain response to flicker and overcome the strict dependence of the standard on the kind of the light source. In this light of fact, this thesis is aimed at presenting an important contribution for a new Flickermeter. An improved visual system model using a physiological parameter that is the mean value of the pupil diameter, has been presented, thus allowing to get a more “objective” representation of the response to flicker. The system used to both generate flicker and measure the pupil diameter has been illustrated along with all the results of several experiments performed on the volunteers. The intent has been to demonstrate that the measurement of that geometrical parameter can give reliable information about the feeling of the human visual system to light flicker.

Kinematic description of the rupture from strong motion data: strategies for a robust inversion

We present a non linear technique to invert strong motion records with the aim of obtaining the final slip and rupture velocity distributions on the fault plane. In this thesis, the ground motion simulation is obtained evaluating the representation integral in the frequency. The Green’s tractions are computed using the discrete wave-number integration technique that provides the full wave-field in a 1D layered propagation medium. The representation integral is computed through a finite elements technique, based on a Delaunay’s triangulation on the fault plane. The rupture velocity is defined on a coarser regular grid and rupture times are computed by integration of the eikonal equation. For the inversion, the slip distribution is parameterized by 2D overlapping Gaussian functions, which can easily relate the spectrum of the possible solutions with the minimum resolvable wavelength, related to source-station distribution and data processing. The inverse problem is solved by a two-step procedure aimed at separating the computation of the rupture velocity from the evaluation of the slip distribution, the latter being a linear problem, when the rupture velocity is fixed. The non-linear step is solved by optimization of an L2 misfit function between synthetic and real seismograms, and solution is searched by the use of the Neighbourhood Algorithm. The conjugate gradient method is used to solve the linear step instead. The developed methodology has been applied to the M7.2, Iwate Nairiku Miyagi, Japan, earthquake. The estimated magnitude seismic moment is 2.6326 dyne∙cm that corresponds to a moment magnitude MW 6.9 while the mean the rupture velocity is 2.0 km/s. A large slip patch extends from the hypocenter to the southern shallow part of the fault plane. A second relatively large slip patch is found in the northern shallow part. Finally, we gave a quantitative estimation of errors associates with the parameters.

Measurement of the muon-induced neutron flux at LNGS with the LVD experiment

In this thesis we describe in detail the Monte Carlo simulation (LVDG4) built to interpret the experimental data collected by LVD and to measure the muon-induced neutron yield in iron and liquid scintillator. A full Monte Carlo simulation, based on the Geant4 (v 9.3) toolkit, has been developed and validation tests have been performed. We used the LVDG4 to determine the active vetoing and the shielding power of LVD. The idea was to evaluate the feasibility to host a dark matter detector in the most internal part, called Core Facility (LVD-CF). The first conclusion is that LVD is a good moderator, but the iron supporting structure produce a great number of neutrons near the core. The second conclusions is that if LVD is used as an active veto for muons, the neutron flux in the LVD-CF is reduced by a factor 50, of the same order of magnitude of the neutron flux in the deepest laboratory of the world, Sudbury. Finally, the muon-induced neutron yield has been measured. In liquid scintillator we found $(3.2 \pm 0.2) \times 10^{-4}$ n/g/cm$^2$, in agreement with previous measurements performed at different depths and with the general trend predicted by theoretical calculations and Monte Carlo simulations. Moreover we present the first measurement, in our knowledge, of the neutron yield in iron: $(1.9 \pm 0.1) \times 10^{-3}$ n/g/cm$^2$. That measurement provides an important check for the MC of neutron production in heavy materials that are often used as shield in low background experiments.

Studio dei raggi cosmici di altissima energia con il progetto EEE

Lo scopo del Progetto Extreme Energy Events (EEE) e` di studiare raggi cosmici di energia estrema, eventi molto rari ma ricchi di informazioni. La grande difficolta` nell'affrontare la fisica dei raggi cosmici di altissima energia risiede nel flusso estremamente basso con cui tali particelle giungono sulla terra. Si utilizzano infatti reti molto estese di rivelatori: le informazioni che si possono ricavare derivano dalla rivelazione delle particelle secondarie prodotte nello sviluppo di sciami estesi di raggi cosmici primari che interagiscono con l'atmosfera terrestre. Il Progetto EEE prevede di dislocare su tutto il territorio italiano un array di telescopi (costituiti da Multi Gap Resistive Plate Chambers) per raggi cosmici secondari. Il lavoro presentato riguarda la simulazione Monte Carlo degli sciami e lo studio delle loro caratteristiche, la simulazione delle prestazioni di griglie di rivelazione differenti ed infine l'analisi dei primi dati raccolti nei telescopi di Bologna, con il conseguente confronto con la simulazione.

Sea Level Variations from Decades to Millennia: A few Case Studies

The main goals of this Ph.D. study are to investigate the regional and global geophysical components related to present polar ice melting and to provide independent cross validation checks of GIA models using both geophysical data detected by satellite mission, and geological observations from far field sites, in order to determine a lower and upper bound of uncertainty of GIA effect. The subject of this Thesis is the sea level change from decades to millennia scale. Within ice2sea collaboration, we developed a Fortran numerical code to analyze the local short-term sea level change and vertical deformation resulting from the loss of ice mass. This method is used to investigate polar regions: Greenland and Antarctica. We have used mass balance based on ICESat data for Greenland ice sheet and a plausible mass balance for Antarctic ice sheet. We have determined the regional and global fingerprint of sea level variations, vertical deformations of the solid surface of the Earth and variations of shape of the geoid for each ice source mentioned above. The coastal areas are affected by the long wavelength component of GIA process. Hence understanding the response of the Earth to loading is crucial in various contexts. Based on the hypothesis that Earth mantle materials obey to a linear rheology, and that the physical parameters of this rheology can be only characterized by their depth dependence, we investigate the Glacial Isostatic Effect upon the far field sites of Mediterranean area using an improved SELEN program. We presented new and revised observations for archaeological fish tanks located along the Tyrrhenian and Adriatic coast of Italy and new RSL for the SE Tunisia. Spatial and temporal variations of the Holocene sea levels studied in central Italy and Tunisia, provided important constraints on the melting history of the major ice sheets.

Metamorfosi temporali e spaziali: dal “Revisore” di Nikolaj Gogol’ alla “Morte accidentale di un anarchico” di Dario Fo.

La tesi si propone di tracciare un confronto tra due autori molto distanti tra loro sia nel tempo che nello spazio: Nikolaj Gogol’ e Dario Fo; in particolare si analizzano le commedie Il Revisore di Gogol’ (1836) e Morte accidentale di un anarchico di Fo (1970). Il nesso tra le due opere è stato individuato dalla critica italiana (Franco Quadri, Ferdinando Taviani, Paolo Puppa). Tuttavia gli spunti interpretativi e comparativi non sono mai stati sviluppati appieno della critica. Nonostante le grandi distanze temporali e spaziali, l’analisi si propone dunque di evidenziare i numerosi motivi di consonanza tra le due opere e i due autori, che peraltro testimoniano anche della grande fortuna arrisa all’estero all’autore ucraino, attraverso la mediazione del teatro russo-sovietico del XX secolo. L’approccio metodologico adottato si fonda sui testi dei formalisti russi, di Lotman, sulla Stilkritik di Aurbach, sugli studi di Frye sulla Bibbia intesa come “grande canone” della letteratura occidentale, e soprattutto sull’analisi della cultura carnevalesca e popolare condotta da Bachtin. Lo studio è indirizzato su alcuni elementi precisi: la trama, lo scambio della personalità, l’impostura, la paura e il riso. Per ciascun di questi elementi è stata svolta l’analisi comparativa delle due opere, situandole in un contesto letterario e culturale che va all’antichità e al Medio Evo, dal quale entrambi gli autori mutuano molte suggestioni.

Genetic engineering and preclinical evaluation of oncolytic herpes simplex viruses retargeted to cancer-specific receptors

Oncolytic virotherapy exploits the ability of viruses to infect and kill cells. It is suitable as treatment for tumors that are not accessible by surgery and/or respond poorly to the current therapeutic approach. HSV is a promising oncolytic agent. It has a large genome size able to accommodate large transgenes and some attenuated oncolytic HSVs (oHSV) are already in clinical trials phase I and II. The aim of this thesis was the generation of HSV-1 retargeted to tumor-specific receptors and detargeted from HSV natural receptors, HVEM and Nectin-1. The retargeting was achieved by inserting a specific single chain antibody (scFv) for the tumor receptor selected inside the HSV glycoprotein gD. In this research three tumor receptors were considered: epidermal growth factor receptor 2 (HER2) overexpressed in 25-30% of breast and ovarian cancers and gliomas, prostate specific membrane antigen (PSMA) expressed in prostate carcinomas and in neovascolature of solid tumors; and epidermal growth factor receptor variant III (EGFRvIII). In vivo studies on HER2 retargeted viruses R-LM113 and R-LM249 have demonstrated their high safety profile. For R-LM249 the antitumor efficacy has been highlighted by target-specific inhibition of the growth of human tumors in models of HER2-positive breast and ovarian cancer in nude mice. In a murine model of HER2-positive glioma in nude mice, R-LM113 was able to significantly increase the survival time of treated mice compared to control. Up to now, PSMA and EGFRvIII viruses (R-LM593 and R-LM613) are only characterized in vitro, confirming the specific retargeting to selected targets. This strategy has proved to be generally applicable to a broad spectrum of receptors for which a single chain antibody is available.

'A Distortion in the mirror of being': verso un modello liquido di personaggio. Da Nabokov alle sperimentazioni contemporanee in ambito slavo

La tesi mira a ridefinire lo statuto del personaggio nell’ambito del self-conscious novel postmoderno, alla luce delle più recenti tendenze narratologiche, con particolare riferimento all’unnatural narratology. Per poter presentare un modello scientificamente valido si è fatto ricorso alla comparazione della produzione letteraria di due macro-aree: quella britannica e quella slava (Russia - Unione Sovietica - e Polonia). Come figura di mediazione tra queste due culture si pone senza dubbio Vladimir V. Nabokov, cardine e personalità di spicco della ricerca. Tra le analisi testuali proposte sono stati presi in considerazione i seguenti autori: Julian Barnes, Vladimir Nabokov, Daniil Charms, Konstantin Vaginov, Andrej Bitov, Saša Sokolov, Bruno Schulz e Tadeusz Kantor.

Essays on Bank Risk

This PhD Thesis is composed of three chapters, each discussing a specific type of risk that banks face. The first chapter talks about Systemic Risk and how banks get exposed to it through the Interbank Funding Market. Exposures in the said market have Systemic Risk implications because the market creates linkages, where the failure of one party can affect the others in the market. By showing that CDS Spreads, as bank risk indicators, are positively related to their Net Interbank Funding Market Exposures, this chapter establishes the above Systemic Risk Implications of Interbank Funding. Meanwhile, the second chapter discusses how banks may handle Illiquidity Risk, defined as the possibility of having sudden funding needs. Illiquidity Risk is embodied in this chapter through Loan Commitments as they oblige banks to lend to its clients, up to a certain amount of funds at any time. This chapter points out that using Securitization as funding facility, could allow the banks to manage this Illiquidity Risk. To make this case, this chapter demonstrates empirically that banks having an increase in Loan Commitments, may experience an increase in risk profile but such can be offset by an accompanying increase in Securitization Activity. Lastly, the third chapter focuses on how banks manage Credit Risk also through Securitization. Securitization has a Credit Risk management property by allowing the offloading of risk. This chapter investigates how banks use such property by looking at the effect of securitization on the banks’ loan portfolios and overall risk and returns. The findings are that securitization is positively related to loan portfolio size and the portfolio share of risky loans, which translates to higher risk and returns. Thus, this chapter points out that Credit Risk management through Securitization may be have been done towards higher risk taking for high returns.

Banking Regulation in Turkey and Russia: An Economic Analysis

The importance of the banks and financial markets relies on the fact that they promote economic efficiency by allocating savings efficiently to profitable investment opportunities.An efficient banking system is a key determinant for the financial stability.The theory of market failure forms the basis for understanding financial regulation.Following the detrimental economic and financial consequences in theaftermath of the crisis, academics and policymakers started to focus their attention on the construction of an appropriate regulatory and supervisory framework of the banking sector. This dissertation aims at understanding the impact of regulations and supervision on banks’ performance focusing on two emerging market economies, Turkey and Russia. It aims at examining the way in which regulations matter for financial stability and banking performance from a law & economics perspective. A review of the theory of banking regulation, particularly as applied to emerging economies, shows that the efficiency of certain solutions regarding banking regulation is open to debate. Therefore, in the context of emerging countries, whether a certain approach is efficient or not will be presented as an empirical question to which this dissertation will try to find an answer.