Characterization of L-type Calcium Channel Binding-Site of a new class of Calcium modulators by a Multidisciplinary approach

Many potential diltiazem related L-VDCC blockers were developed using a multidisciplinary approach. This current study was to investigate and compare diltiazem with to the newly developed compounds by mouse Langendorff-perfused heart, Ca2+-transient and on recombinant L-VDCC. Twenty particular compounds were selected by the ligand-based virtual screening procedure (LBVS). From these compounds, five of them (5b, M2, M7, M8 and P1) showed a potent and selective inotropic activity on guinea-pig left atria driven 1 Hz. Further assays displayed an interesting negative inotropic effect of M2, M8, P1 and M7 on guinea pig isolated left papillary muscle driven at 1 Hz, a relevant vasorelaxant activity of 5b, M2, M7, M8 and P1 on K+-depolarized guinea-pig ileum longitudinal smooth muscle and a significant inhibition of contraction of 5b, M2, M8 and P1 on carbachol stimulated ileum longitudinal smooth muscle. Wild-type human heart and rabbit lung α1 subunits were expressed (combined with the regulatory α2δ and β3 subunits) in Xenopus Leavis oocytes using a two-electrode voltage clamp technique. Diltiazem is a benzothiazepine Ca2+ channel blocker used clinically for its antihypertensive and antiarrhythmic effects. Previous radioligand binding assays revealed a complex interaction with the benzothiazepine binding site for M2, M7 and M8. (Carosati E. et al. J. Med Chem. 2006, 49; 5206). In agreement with this findings, the relative order of increased rates of contraction and relaxation at lower concentrations s(≤10-6M) in unpaced hearts was M7>M2>M8>P1. Similar increases in Ca2+ transient were observed in cardiomyocytes. Diltiazem showed negative inotropic effects whereas 5b had no significant effect. Diltiazem blocks Ca2+current in a use-dependent manner and facilitates the channel by accelerating the inactivation and decelerating the recovery from inactivation. In contrast to diltiazem, the new analogs had no pronounced use-dependence. Application of 100 μM M8, M2 showed ~ 10% tonic block; in addition, M8, M2 and P1 shifted the steady state inactivation in hyperpolarized direction and the current inactivation time was significantly decreased compared with control (219.6 ± 11.5 ms, 226 ± 14.5 vs. 269 ± 12.9 vs. 199.28 ± 8.19 ms). Contrary to diltiazem, the recovery from the block by M8 and M2 was comparable to control. Only P1 showed a significantly decrease of the time for the recovery from inactivation. All of the compounds displayed the same sensitivity on the Ca2+ channel rabbit lung α1 except P1. Taken together, these findings suggest that M8, M2 and P1 might directly decrease the binding affinity or allow rapid dissociation from the benzothiazepine binding site.

Valutazione dell'efficacia delle procedure di Pushover convenzionali, modali ed adattive applicate a telai piani in c.a. regolari ed irregolari in elevazione.

The research performed during the PhD and presented in this thesis, allowed to make judgments on pushover analysis method about its application in evaluating the correct structural seismic response. In this sense, the extensive critical review of existing pushover procedures (illustrated in chapter 1) outlined their major issues related to assumptions and to hypothesis made in the application of the method. Therefore, with the purpose of evaluate the effectiveness of pushover procedures, a wide numerical investigation have been performed. In particular the attention has been focused on the structural irregularity on elevation, on the choice of the load vector and on its updating criteria. In the study eight pushover procedures have been considered, of which four are conventional type, one is multi-modal, and three are adaptive. The evaluation of their effectiveness in the identification of the correct dynamic structural response, has been done by performing several dynamic and static non-linear analysis on eight RC frames, characterized by different proprieties in terms of regularity in elevation. The comparisons of static and dynamic results have then permitted to evaluate the examined pushover procedures and to identify the expected margin of error by using each of them. Both on base shear-top displacement curves and on considered storey parameters, the best agreement with the dynamic response has been noticed on Multi-Modal Pushover procedure. Therefore the attention has been focused on Displacement-based Adative Pushover, coming to define for it an improvement strategy, and on modal combination rules, advancing an innovative method based on a quadratic combination of the modal shapes (QMC). This latter has been implemented in a conventional pushover procedure, whose results have been compared with those obtained by other multi-modal procedures. The development of research on pushover analysis is very important because the objective is to come to the definition of a simple, effective and reliable analysis method, indispensable tool in the seismic evaluation of new or existing structures.

The potential of the 3-UPU translational parallel manipulator and a procedure to select the best architecture

The 3-UPU three degrees of freedom fully parallel manipulator, where U and P are for universal and prismatic pair respectively, is a very well known manipulator that can provide the platform with three degrees of freedom of pure translation, pure rotation or mixed translation and rotation with respect to the base, according to the relative directions of the revolute pair axes (each universal pair comprises two revolute pairs with intersecting and perpendicular axes). In particular, pure translational parallel 3-UPU manipulators (3-UPU TPMs) received great attention. Many studies have been reported in the literature on singularities, workspace, and joint clearance influence on the platform accuracy of this manipulator. However, much work has still to be done to reveal all the features this topology can offer to the designer when different architecture, i.e. different geometry are considered. Therefore, this dissertation will focus on this type of the 3-UPU manipulators. The first part of the dissertation presents six new architectures of the 3-UPU TPMs which offer interesting features to the designer. In the second part, a procedure is presented which is based on some indexes, in order to allows the designer to select the best architecture of the 3-UPU TPMs for a given task. Four indexes are proposed as stiffness, clearance, singularity and size of the manipulator in order to apply the procedure.

Oltre il limite invalicabile. Procedure, piani, progetti sostenibili per le aree militari dismissibili di Piacenza

The research project concerns the restoration of military real estate, starting from the procedures in progress in Italy related to dismission, with particular emphasis for Piacenza and the case study of Caserme Bixio and Nicolai (Comparto Nord/San Sisto-Cittadella). The work is aimed at defining the relationship between urban planning and rehabilitation of dismissed military areas, in order to create a synergic effort capable to face the transformation of these sites and their integration into the urban tissue. The results obtained from the various recordings performed in Italy and in USA during the preparation of this thesis may help to find a new possible scenario for the Comparto Nord and its historic buildings.

New mechanisms for modelling the motion of the human ankle complex

The relevance of human joint models was shown in the literature. In particular, the great importance of models for the joint passive motion simulation (i.e. motion under virtually unloaded conditions) was outlined. They clarify the role played by the principal anatomical structures of the articulation, enhancing the comprehension of surgical treatments, and in particular the design of total ankle replacement and ligament reconstruction. Equivalent rigid link mechanisms proved to be an efficient tool for an accurate simulation of the joint passive motion. This thesis focuses on the ankle complex (i.e. the anatomical structure composed of the tibiotalar and the subtalar joints), which has a considerable role in human locomotion. The lack of interpreting models of this articulation and the poor results of total ankle replacement arthroplasty have strongly suggested devising new mathematical models capable of reproducing the restraining function of each structure of the joint and of replicating the relative motion of the bones which constitute the joint itself. In this contest, novel equivalent mechanisms are proposed for modelling the ankle passive motion. Their geometry is based on the joint’s anatomical structures. In particular, the role of the main ligaments of the articulation is investigated under passive conditions by means of nine 5-5 fully parallel mechanisms. Based on this investigation, a one-DOF spatial mechanism is developed for modelling the passive motion of the lower leg. The model considers many passive structures constituting the articulation, overcoming the limitations of previous models which took into account few anatomical elements of the ankle complex. All the models have been identified from experimental data by means of optimization procedure. Then, the simulated motions have been compared to the experimental one, in order to show the efficiency of the approach and thus to deduce the role of each anatomical structure in the ankle kinematic behavior.

Melanocytes: a new potential tool to study and monitoring Duchenne muscular dystrophy

Dystrophin is a subsarcolemmal protein critical for the integrity of muscle fibers by linking the actin cytoskeleton to the extracellular matrix via the dystroglycan complex. It is reported that dystroglycans are also localized in the skin, at dermal-epidermal junction. Here we show that epidermal melanocytes express dystrophin at the interface with the basement membrane. The full-length muscle isoform mDp427 was clearly detectable in epidermis and in melanocyte cultures as assessed by RNA and western blot analysis. Dystrophin was absent in Duchenne Muscular Dystrophy (DMD) patients melanocytes, and the ultrastructural analysis revealed mitochondrial alterations, similar to those occurring in myoblasts from the same patients. Interestingly, mitochondrial dysfunction of DMD melanocytes reflected the alterations identified in dystrophin-deficient muscle cells. In fact, mitochondria of melanocytes from DMD patients accumulated tetramethylrhodamine methyl ester but, on the contrary of control donor, mitochondria of DMD patients readily depolarized upon the addition of oligomycin, suggesting either that they are maintaining the membrane potential at the expense of glycolytic ATP, or that they are affected by a latent dysfunction unmasked by inhibition of the ATP synthase. Melanocyte cultures can be easily obtained by conventional skin biopsies, less invasive procedure than muscular biopsy, so that they may represent an alternative cellular model to myoblast for studying and monitoring dystrophinopathies also in response to pharmacological treatments.

Lymphogranuloma venereum: a new face for an old disease. Our experience in the Bologna area.

Lymphogranuloma venereum (LGV) is a sexual transmitted infection due to Clamydia trachomatis biovar L, endemic in part of Africa, Asia, South America and the Caribbean, but rare in industrialized countries up to 10 years ago. In 2003, a cluster of cases of LGV among men who have sex with men (MSM) was reported in Rotterdam. Since then, several reports of LGV have been reported in the largest cities in Europe, the United States and Australia. They have usually occurred with an anorectal syndrome. The purpose of this study is to summarize the expertise provided by the international literature about the new LGV outbreaks and to offer the first data collected on the presence of this disease in the Bologna area. In fact, we examine 5 cases of LGV proctitis diagnosed and treated at the Clinic of Sexually Transmitted Disease (STD) of the Dermatology Section at Sant’Orsola-Malpighi Hospital, Bologna. Particular attention will be paid to the laboratory method that allows identification and typing of the microorganism C. trachomatis serovar L1, L2, L3, leading to an etiologic diagnosis of certainty. The diagnosed cases of LGV will be described and compared with the international literature, trying to assess the risk factors, the most effective diagnostic and therapeutic procedure and the best approach to the patient.

New regularization method for EXAFS analysis: Application to uranium and plutonium sorption onto kaolinite

Die Röntgenabsorptionsspektroskopie (Extended X-ray absorption fine structure (EXAFS) spectroscopy) ist eine wichtige Methode zur Speziation von Schwermetallen in einem weiten Bereich von umweltrelevanten Systemen. Um Strukturparameter wie Koordinationszahl, Atomabstand und Debye-Waller Faktoren für die nächsten Nachbarn eines absorbierenden Atoms zu bestimmen, ist es für experimentelle EXAFS-Spektren üblich, unter Verwendung von Modellstrukturen einen „Least-Squares-Fit“ durchzuführen. Oft können verschiedene Modellstrukturen mit völlig unterschiedlicher chemischer Bedeutung die experimentellen EXAFS-Daten gleich gut beschreiben. Als gute Alternative zum konventionellen Kurven-Fit bietet sich das modifizierte Tikhonov-Regularisationsverfahren an. Ergänzend zur Tikhonov-Standardvariationsmethode enthält der in dieser Arbeit vorgestellte Algorithmus zwei weitere Schritte, nämlich die Anwendung des „Method of Separating Functionals“ und ein Iterationsverfahren mit Filtration im realen Raum. Um das modifizierte Tikhonov-Regularisationsverfahren zu testen und zu bestätigen wurden sowohl simulierte als auch experimentell gemessene EXAFS-Spektren einer kristallinen U(VI)-Verbindung mit bekannter Struktur, nämlich Soddyit (UO2)2SiO4 x 2H2O, untersucht. Die Leistungsfähigkeit dieser neuen Methode zur Auswertung von EXAFS-Spektren wird durch ihre Anwendung auf die Analyse von Proben mit unbekannter Struktur gezeigt, wie sie bei der Sorption von U(VI) bzw. von Pu(III)/Pu(IV) an Kaolinit auftreten. Ziel der Dissertation war es, die immer noch nicht voll ausgeschöpften Möglichkeiten des modifizierten Tikhonov-Regularisationsverfahrens für die Auswertung von EXAFS-Spektren aufzuzeigen. Die Ergebnisse lassen sich in zwei Kategorien einteilen. Die erste beinhaltet die Entwicklung des Tikhonov-Regularisationsverfahrens für die Analyse von EXAFS-Spektren von Mehrkomponentensystemen, insbesondere die Wahl bestimmter Regularisationsparameter und den Einfluss von Mehrfachstreuung, experimentell bedingtem Rauschen, etc. auf die Strukturparameter. Der zweite Teil beinhaltet die Speziation von sorbiertem U(VI) und Pu(III)/Pu(IV) an Kaolinit, basierend auf experimentellen EXAFS-Spektren, die mit Hilfe des modifizierten Tikhonov-Regularisationsverfahren ausgewertet und mit Hilfe konventioneller EXAFS-Analyse durch „Least-Squares-Fit“ bestätigt wurden.

Dynamic resolution of alpha-substituted dihydrocinnamic aldehydes. A new asymmetric synthesis of pharmaceutically important amine building blocks.

Crystallization-induced diastereoisomer transformation (CIDT) was successfully employed in the enantioselective synthesis of 2-alkyl-3-aryl-propan-1-amines. These products are seen as potentially useful building blocks in the field of asymmetric organic chemistry, notably for pharmaceutically relevant compounds. The procedure was based on a recently reported protocol for deracemization of dihydrocinnamic aldehydes in which enantiomerically enriched 1-(amino(phenyl)methyl)naphthalen-2-ol (Betti base) is employed as a resolving agent. Additionally, fenpropimorph, a biologically active substance which contains the 2-alkyl-3-aryl-propan-1-amine moiety was synthetized, as an attempt to assess the usefulness of the enantiomerically enriched amines.

A new double laser pulse pumping scheme for transient collisionally excited plasma soft X-ray lasers

Within this thesis a new double laser pulse pumping scheme for plasma-based, transient collisionally excited soft x-ray lasers (SXRL) was developed, characterized and utilized for applications. SXRL operations from ~50 up to ~200 electron volt were demonstrated applying this concept. As a central technical tool, a special Mach-Zehnder interferometer in the chirped pulse amplification (CPA) laser front-end was developed for the generation of fully controllable double-pulses to optimally pump SXRLs.rnThis Mach-Zehnder device is fully controllable and enables the creation of two CPA pulses of different pulse duration and variable energy balance with an adjustable time delay. Besides the SXRL pumping, the double-pulse configuration was applied to determine the B-integral in the CPA laser system by amplifying short pulse replica in the system, followed by an analysis in the time domain. The measurement of B-integral values in the 0.1 to 1.5 radian range, only limited by the reachable laser parameters, proved to be a promising tool to characterize nonlinear effects in the CPA laser systems.rnContributing to the issue of SXRL pumping, the double-pulse was configured to optimally produce the gain medium of the SXRL amplification. The focusing geometry of the two collinear pulses under the same grazing incidence angle on the target, significantly improved the generation of the active plasma medium. On one hand the effect was induced by the intrinsically guaranteed exact overlap of the two pulses on the target, and on the other hand by the grazing incidence pre-pulse plasma generation, which allows for a SXRL operation at higher electron densities, enabling higher gain in longer wavelength SXRLs and higher efficiency at shorter wavelength SXRLs. The observation of gain enhancement was confirmed by plasma hydrodynamic simulations.rnThe first introduction of double short-pulse single-beam grazing incidence pumping for SXRL pumping below 20 nanometer at the laser facility PHELIX in Darmstadt (Germany), resulted in a reliable operation of a nickel-like palladium SXRL at 14.7 nanometer with a pump energy threshold strongly reduced to less than 500 millijoule. With the adaptation of the concept, namely double-pulse single-beam grazing incidence pumping (DGRIP) and the transfer of this technology to the laser facility LASERIX in Palaiseau (France), improved efficiency and stability of table-top high-repetition soft x-ray lasers in the wavelength region below 20 nanometer was demonstrated. With a total pump laser energy below 1 joule the target, 2 mircojoule of nickel-like molybdenum soft x-ray laser emission at 18.9 nanometer was obtained at 10 hertz repetition rate, proving the attractiveness for high average power operation. An easy and rapid alignment procedure fulfilled the requirements for a sophisticated installation, and the highly stable output satisfied the need for a reliable strong SXRL source. The qualities of the DGRIP scheme were confirmed in an irradiation operation on user samples with over 50.000 shots corresponding to a deposited energy of ~ 50 millijoule.rnThe generation of double-pulses with high energies up to ~120 joule enabled the transfer to shorter wavelength SXRL operation at the laser facility PHELIX. The application of DGRIP proved to be a simple and efficient method for the generation of soft x-ray lasers below 10 nanometer. Nickel-like samarium soft x-ray lasing at 7.3 nanometer was achieved at a low total pump energy threshold of 36 joule, which confirmed the suitability of the applied pumping scheme. A reliable and stable SXRL operation was demonstrated, due to the single-beam pumping geometry despite the large optical apertures. The soft x-ray lasing of nickel-like samarium was an important milestone for the feasibility of applying the pumping scheme also for higher pumping pulse energies, which are necessary to obtain soft x-ray laser wavelengths in the water window. The reduction of the total pump energy below 40 joule for 7.3 nanometer short wavelength lasing now fulfilled the requirement for the installation at the high-repetition rate operation laser facility LASERIX.rn

Development Of New Toolkits For Orbit Determination Codes For Precise Radio Tracking Experiments

This thesis describes the developments of new models and toolkits for the orbit determination codes to support and improve the precise radio tracking experiments of the Cassini-Huygens mission, an interplanetary mission to study the Saturn system. The core of the orbit determination process is the comparison between observed observables and computed observables. Disturbances in either the observed or computed observables degrades the orbit determination process. Chapter 2 describes a detailed study of the numerical errors in the Doppler observables computed by NASA's ODP and MONTE, and ESA's AMFIN. A mathematical model of the numerical noise was developed and successfully validated analyzing against the Doppler observables computed by the ODP and MONTE, with typical relative errors smaller than 10%. The numerical noise proved to be, in general, an important source of noise in the orbit determination process and, in some conditions, it may becomes the dominant noise source. Three different approaches to reduce the numerical noise were proposed. Chapter 3 describes the development of the multiarc library, which allows to perform a multi-arc orbit determination with MONTE. The library was developed during the analysis of the Cassini radio science gravity experiments of the Saturn's satellite Rhea. Chapter 4 presents the estimation of the Rhea's gravity field obtained from a joint multi-arc analysis of Cassini R1 and R4 fly-bys, describing in details the spacecraft dynamical model used, the data selection and calibration procedure, and the analysis method followed. In particular, the approach of estimating the full unconstrained quadrupole gravity field was followed, obtaining a solution statistically not compatible with the condition of hydrostatic equilibrium. The solution proved to be stable and reliable. The normalized moment of inertia is in the range 0.37-0.4 indicating that Rhea's may be almost homogeneous, or at least characterized by a small degree of differentiation.

New and More Sustainable Processes for the Synthesis of Phenolics: 2-phenoxyethanol and hydroxytyrosol

The research work has dealt with the study of new catalytic processes for the synthesis of fine chemicals belonging to the class of phenolics, namely 2-phenoxyethanol and hydroxytyrosol. The two synthetic procedures investigated have the advantages of being much closer to the Green Chemistry principles than those currently used industrially. In both cases, the challenge was that of finding catalysts and methods which led to the production of less waste, and used less hazardous chemicals, safer solvents, and reusable heterogeneous catalysts. In the case of 2-phenoxyethanol, the process investigated involves the use of ethylene carbonate (EC) as the reactant for phenol O-hydroxyethylation, in place of ethylene oxide. Besides being a safer reactant, the major advantage of using EC in the new synthesis is the better selectivity to the desired product achieved. Moreover, the solid catalyst based on Na-mordenite was fully recyclable. The reaction mechanism and the effect of the Si/Al ratio in the mordenite were investigated. In the case of hydroxytyrosol, which is one of the most powerful natural antioxidants, a new synthetic procedure was investigated; in fact, the method currently employed, the hydrolysis of oleuropein, an ester extracted from the waste water processing of the olive, makes use of large amounts of organic solvents (hexane, ethyl acetate), and involves several expensive steps of purification. The synthesis procedure set up involves first the reaction between catechol and 2,2-dimethoxyacetaldehyde, followed by the one-pot reduction of the intermediate to give the desired product. Both steps were optimized, in terms of catalyst used, and of reaction conditions, that allowed to reach ca 70% yield in each step. The reaction mechanism was investigated and elucidated. During a 3-month period spent at the University of Valencia (with Prof. A. Corma’s group), a process for the production of diesel additives (2,5-bis(propoxymethyl)furan) from fructose has been investigated.

A New Test Rig for In-Vitro Evaluation of the Knee Joint Behaviour

The evaluation of the knee joint behavior is fundamental in many applications, such as joint modeling, prosthesis and orthosis design. In-vitro tests are important in order to analyse knee behavior when simulating various loading conditions and studying physiology of the joint. A new test rig for in-vitro evaluation of the knee joint behavior is presented in this paper. It represents the evolution of a previously proposed rig, designed to overcome its principal limitations and to improve its performances. The design procedure and the adopted solution in order to satisfy the specifications are presented here. Thanks to its 6-6 Gough-Stewart parallel manipulator loading system, the rig replicates general loading conditions, like daily actions or clinical tests, on the specimen in a wide range of flexion angles. The restraining actions of knee muscles can be simulated when active actions are simulated. The joint motion in response to the applied loads, guided by passive articular structures and muscles, is permitted by the characteristics of the loading system which is force controlled. The new test rig guarantees visibility so that motion can be measured by an optoelectronic system. Furthermore, the control system of the new test rig allows the estimation of the contribution of the principal leg muscles in guaranteeing the equilibrium of the joint by the system for muscle simulation. Accuracy in positioning is guaranteed by the designed tibia and femur fixation systems,which allow unmounting and remounting the specimen in the same pose. The test rig presented in this paper permits the analysis of the behavior of the knee joint and comparative analysis on the same specimen before and after surgery, in a way to assess the goodness of prostheses or surgical treatments.

Multimodal Registration Procedure for the Initial Spatial Alignment of a Retinal Video Sequence to a Retinal Composite Image

Accurate placement of lesions is crucial for the effectiveness and safety of a retinal laser photocoagulation treatment. Computer assistance provides the capability for improvements to treatment accuracy and execution time. The idea is to use video frames acquired from a scanning digital ophthalmoscope (SDO) to compensate for retinal motion during laser treatment. This paper presents a method for the multimodal registration of the initial frame from an SDO retinal video sequence to a retinal composite image, which may contain a treatment plan. The retinal registration procedure comprises the following steps: 1) detection of vessel centerline points and identification of the optic disc; 2) prealignment of the video frame and the composite image based on optic disc parameters; and 3) iterative matching of the detected vessel centerline points in expanding matching regions. This registration algorithm was designed for the initialization of a real-time registration procedure that registers the subsequent video frames to the composite image. The algorithm demonstrated its capability to register various pairs of SDO video frames and composite images acquired from patients.

Procedural and mid-term results in patients with aortic stenosis treated with implantation of 2 (in-series) CoreValve prostheses in 1 procedure

This study sought to assess post-procedural and mid-term outcome of patients, in which a second "in-series" CoreValve prosthesis (Medtronic, Minneapolis, Minnesota) was implanted during the same procedure.