Molecular cloning, heterologous expression and characterization of Strictosidine Glucosidase from Rauvolfia serpentina cell suspension cultures

Die vorliegende Arbeit hatte zum Ziel, die enzymatische Deglucosylierung von Strictosidin in Zellsuspensionskulturen von Rauvolfia serpentina zu charakterisieren.Ein Verfahren zur Isolierung und Reinigung von Strictosidin aus pflanzlicher Zellkulturen wurde entwickelt. Zwei somatische Hybridzellkulturen zwischen R. serpentina und Rhazya stricta wurden als potenzielle Quelle dieses Glucoalkaloides untersucht. Der Sekundärstoffwechsel der pflanzlichen Zellen wurde mit Methyljasmonat induziert und 15 Stoffe wurden identifiziert, u. a. das neue Indolalkaloid 3-Oxo-rhazinilam. Die Gehaltsänderung von 7 Indolalkaloiden nach Behandlung mit Methyljasmonat wurde untersucht.Deglucosylierung von Strictisidin bei in E. coli exprimierter Raucaffricin Glucosidase wurde detektiert.Die Strictosidin Glucosidase kodierende cDNA wurde aus R. serpentina Zellsuspensionskulturen cloniert und in E. coli exprimiert. Das Enzyme wurde mit Hilfe des Inteintages gereinigt und seine Eigenschaften wurden untersucht, u. a. optimale Temperatur und pH Wert und Substratspezifität.Die Produkte von der enzymatischen Strictosidinhydrolyse wurden als Cathenamin (unter normalen Bedingungen) und Sitsirikin und Isositsirikin (im Gegenwart von Reduktoren) identifiziert. Das neue Indolalkaloid 3-Isocorreantin A wurde nach der enzymatischen Deglucosylierung von Dolichantosid (Nß-Methylstrictosidin) gebildet.

Pushover analysis of an existing reinforced concrete bridge:Jamboree Road Overcrossing in Irvine, California

The work for the present thesis started in California, during my semester as an exchange student overseas. California is known worldwide for its seismicity and its effort in the earthquake engineering research field. For this reason, I immediately found interesting the Structural Dynamics Professor, Maria Q. Feng's proposal, to work on a pushover analysis of the existing Jamboree Road Overcrossing bridge. Concrete is a popular building material in California, and for the most part, it serves its functions well. However, concrete is inherently brittle and performs poorly during earthquakes if not reinforced properly. The San Fernando Earthquake of 1971 dramatically demonstrated this characteristic. Shortly thereafter, code writers revised the design provisions for new concrete buildings so to provide adequate ductility to resist strong ground shaking. There remain, nonetheless, millions of square feet of non-ductile concrete buildings in California. The purpose of this work is to perform a Pushover Analysis and compare the results with those of a Nonlinear Time-History Analysis of an existing bridge, located in Southern California. The analyses have been executed through the software OpenSees, the Open System for Earthquake Engineering Simulation. The bridge Jamboree Road Overcrossing is classified as a Standard Ordinary Bridge. In fact, the JRO is a typical three-span continuous cast-in-place prestressed post-tension box-girder. The total length of the bridge is 366 ft., and the height of the two bents are respectively 26,41 ft. and 28,41 ft.. Both the Pushover Analysis and the Nonlinear Time-History Analysis require the use of a model that takes into account for the nonlinearities of the system. In fact, in order to execute nonlinear analyses of highway bridges it is essential to incorporate an accurate model of the material behavior. It has been observed that, after the occurrence of destructive earthquakes, one of the most damaged elements on highway bridges is a column. To evaluate the performance of bridge columns during seismic events an adequate model of the column must be incorporated. Part of the work of the present thesis is, in fact, dedicated to the modeling of bents. Different types of nonlinear element have been studied and modeled, with emphasis on the plasticity zone length determination and location. Furthermore, different models for concrete and steel materials have been considered, and the selection of the parameters that define the constitutive laws of the different materials have been accurate. The work is structured into four chapters, to follow a brief overview of the content. The first chapter introduces the concepts related to capacity design, as the actual philosophy of seismic design. Furthermore, nonlinear analyses both static, pushover, and dynamic, time-history, are presented. The final paragraph concludes with a short description on how to determine the seismic demand at a specific site, according to the latest design criteria in California. The second chapter deals with the formulation of force-based finite elements and the issues regarding the objectivity of the response in nonlinear field. Both concentrated and distributed plasticity elements are discussed into detail. The third chapter presents the existing structure, the software used OpenSees, and the modeling assumptions and issues. The creation of the nonlinear model represents a central part in this work. Nonlinear material constitutive laws, for concrete and reinforcing steel, are discussed into detail; as well as the different scenarios employed in the columns modeling. Finally, the results of the pushover analysis are presented in chapter four. Capacity curves are examined for the different model scenarios used, and failure modes of concrete and steel are discussed. Capacity curve is converted into capacity spectrum and intersected with the design spectrum. In the last paragraph, the results of nonlinear time-history analyses are compared to those of pushover analysis.

Comparison between analytical and numerical methods for the assessment of masonry arch bridges: Case study of Clemente Bridge on Savio river

This research has focused on the study of the behavior and of the collapse of masonry arch bridges. The latest decades have seen an increasing interest in this structural type, that is still present and in use, despite the passage of time and the variation of the transport means. Several strategies have been developed during the time to simulate the response of this type of structures, although even today there is no generally accepted standard one for assessment of masonry arch bridges. The aim of this thesis is to compare the principal analytical and numerical methods existing in literature on case studies, trying to highlight values and weaknesses. The methods taken in exam are mainly three: i) the Thrust Line Analysis Method; ii) the Mechanism Method; iii) the Finite Element Methods. The Thrust Line Analysis Method and the Mechanism Method are analytical methods and derived from two of the fundamental theorems of the Plastic Analysis, while the Finite Element Method is a numerical method, that uses different strategies of discretization to analyze the structure. Every method is applied to the case study through computer-based representations, that allow a friendly-use application of the principles explained. A particular closed-form approach based on an elasto-plastic material model and developed by some Belgian researchers is also studied. To compare the three methods, two different case study have been analyzed: i) a generic masonry arch bridge with a single span; ii) a real masonry arch bridge, the Clemente Bridge, built on Savio River in Cesena. In the analyses performed, all the models are two-dimensional in order to have results comparable between the different methods taken in exam. The different methods have been compared with each other in terms of collapse load and of hinge positions.

On the identification of bridge decks aeroelastic coefficients: the covariance block hankel matrix method

Il seguente elaborato si concentra sull'identifi�cazione strutturale di sistemi soggetti a sollecitazioni aeroelastiche e nello speci�fico l'attenzione viene rivolta ad impalcati da ponte. Si analizzano i concetti principali caratterizzanti il campo dell'aeroelasticità indagando i fattori dominanti che entrano in gioco sul piano teorico. In seguito, si considera il metodo di identifi�cazione strutturale chiamato Covariance Block Hankel Matrix (CBHM) utilizzato come strumento di derivazione dei coeffi�cienti aeroelastici propri della struttura. Infi�ne, si indaga il comportamento di questo metodo di identi�ficazione al variare di una serie di parametri chiave e all'interno di diversi scenari, visionando risultati ottenuti tramite una serie di test eff�ettuati per provare l'a�dattabilità del metodo stesso al variare delle condizioni che caratterizzano il sistema.

Aereodynamics of dry helically-filleted bridge cables

Dry limited amplitude vibrations flow-transition induced vibrations were experienced on a helically-filleted tube, in a previous study performed by Kleissl and Georgakis (2012). These vibrations have never been reported in previous studies. A deep study on the same inclined-yawed cable configuration has been performed, in order to investigate and further understand the nature of these vibrations. The investigation has been carried out through passive-dynamic wind tunnel tests in the Climatic Wind Tunnel at FORCE Technology, Kgs. Lyngby, Denmark. The results are carried out in terms of aerodynamic damping and peak to peak amplitude at different flow velocities and different boundary conditions. The latter are done by testing the model with and without the spray system installed in the wind tunnel cross section, in order to understand and evaluate the influence of the spray system on the start of the vibrations mechanism and on the flow turbulence. The gained experiences are finally presented for the use in future testing activities with the purpose of improving the performance of passive-dynamic tests.

The posterior parietal cortex: a bridge between vision and action

The present work takes into account three posterior parietal areas, V6, V6A, and PEc, all operating on different subsets of signals (visual, somatic, motor). The work focuses on the study of their functional properties, to better understand their respective contribution in the neuronal circuits that make possible the interactions between subject and external environment. In the caudalmost pole of parietal lobe there is area V6. Functional data suggest that this area is related to the encoding of both objects motion and ego-motion. However, the sensitivity of V6 neurons to optic flow stimulations has been tested only in human fMRI experiments. Here we addressed this issue by applying on monkey the same experimental protocol used in human studies. The visual stimulation obtained with the Flow Fields stimulus was the most effective and powerful to activate area V6 in monkey, further strengthening this homology between the two primates. The neighboring areas, V6A and PEc, show different cytoarchitecture and connectivity profiles, but are both involved in the control of reaches. We studied the sensory responses present in these areas, and directly compared these.. We also studied the motor related discharges of PEc neurons during reaching movements in 3D space comparing also the direction and depth tuning of PEc cells with those of V6A. The results show that area PEc and V6A share several functional properties. Area PEc, unlike V6A, contains a richer and more complex somatosensory input, and a poorer, although complex visual one. Differences emerged also comparing the motor-related properties for reaches in depth: the incidence of depth modulations in PEc and the temporal pattern of modulation for depth and direction allow to delineate a trend among the two parietal visuomotor areas.

Balloon Aortic Valvuloplasty as Bridge-to-Decision in High Risk Patients with Severe Aortic Valve Stenosis

Background. A sizable group of patients with symptomatic aortic stenosis (AS) can undergo neither surgical aortic valve replacement (AVR) nor transcatheter aortic valve implantation (TAVI) because of clinical contraindications. The aim of this study was to assess the potential role of balloon aortic valvuloplasty (BAV) as a “bridge-to-decision” in selected patients with severe AS and potentially reversible contraindications to definitive treatment. Methods. We retrospectively enrolled 645 patients who underwent first BAV at our Institution between July 2007 and December 2012. Of these, the 202 patients (31.2%) who underwent BAV as bridge-to-decision (BTD) requiring clinical re-evaluation represented our study population. BTD patients were further subdivided in 5 groups: low left ventricular ejection fraction; mitral regurgitation grade ≥3; frailty; hemodynamic instability; comorbidity. The main objective of the study was to evaluate how BAV influenced the final treatment strategy in the whole BTD group and in its single specific subgroups. Results. Mean logistic EuroSCORE was 23.5±15.3%, mean age was 81±7 years. Mean transaortic gradient decreased from 47±17 mmHg to 33±14 mmHg. Of the 193 patients with BTD-BAV who received a second heart team evaluation, 72.5% were finally deemed eligible for definitive treatment (25.4%for AVR; 47.2% for TAVI): respectively, 96.7% of patients with left ventricular ejection fraction recovery; 70.5% of patients with mitral regurgitation reduction; 75.7% of patients who underwent BAV in clinical hemodynamic instability; 69.2% of frail patients and 68% of patients who presented relevant comorbidities. 27.5% of the study population was deemed ineligible for definitive treatment and treated with standard therapy/repeated BAV. In-hospital mortality was 4.5%, cerebrovascular accident occurred in 1% and overall vascular complications were 4% (0.5% major; 3.5% minor). Conclusions. Balloon aortic valvuloplasty should be considered as bridge-to-decision in high-risk patients with severe aortic stenosis who cannot be immediate candidates for definitive percutaneous or surgical treatment.

Investigation of different shellac grades and improvement of release from air suspension coated pellets

Shellac is the purified product of the natural polymer Lac. Shellac types, from different origins and with different ages, all purified by the solvent extraction process were compared in this study. Their physicochemical properties acid value, glass transition temperatures, color numbers and molecular sizes were determined. Metoprolol tartrate pellets were coated by air suspension coating with these different grades of shellac. Two coating levels 20% w/w and 25% w/w were applied and then subjected to in vitro dissolution testing. Enteric resistance was achieved for all tested brands for the two coating levels. At pH 6.8, 7.2 and 7.4, significant variations were obvious between the brands. rnMoreover the molecular size of shellac has a pronounced effect in that shellac types with larger molecular size show a higher and faster release than others, while the one with the smaller molecular size show the opposite effect on the release of metoprolol.rnIn this study commercially available ready for use aqueous shellac solutions (SSB AQUAGOLD), which are based on shellac SSB 57 (Dewaxed Orange Shellac, Bysakhi-Ber type refined in a solvent extraction process), with different manufacturing dates were used. rnTo improve the enteric coating properties of films from aqueous shellac solutions, different aqueous polymeric solutions of hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), carboyxmethyl cellulose (CMC), gum arabic and polysaccharides (Pullulan®) were used. These water soluble polymers will act as pore formers to enhance drug release from pellets coated with the combination of shellac and these polymers. The influence of these polymers on the gloss of the shellac films, mechanical properties of the films and drug release from metoprolol tartrate pellets were studied.rnThe potential of ethanol to alter the rate of drug release from shellac coated pellets was assessed by using a modified in vitro dose dumping in alcohol (DDA) method and the test concluded that shellac coated dosage forms can be co-administered with alcohol beverages containing ≤ 5% with no effect of alcohol on the shellac coat.rnPellets coated with shellac sodium salts, showed higher release rates than pellets coated with shellac as ammonium salt forms. rn

Life cycle assessment in construction field: A life cycle cost analysis of reinforcement concrete bridge

The present work is included in the context of the assessment of sustainability in the construction field and is aimed at estimating and analyzing life cycle cost of the existing reinforced concrete bridge “Viadotto delle Capre” during its entire life. This was accomplished by a comprehensive data collection and results evaluation. In detail, the economic analysis of the project is performed. The work has investigated possible design alternatives for maintenance/rehabilitation and end-of-life operations, when structural, functional, economic and also environmental requirements have to be fulfilled. In detail, the economic impact of different design options for the given reinforced concrete bridge have been assessed, whereupon the most economically, structurally and environmentally efficient scenario was chosen. The Integrated Life-Cycle Analysis procedure and Environmental Impact Assessment were also discussed in this work. The scope of this thesis is to illustrate that Life Cycle Cost analysis as part of Life Cycle Assessment approach could be effectively used to drive the design and management strategy of new and existing structures. The final objective of this contribution is to show how an economic analysis can influence decision-making in the definition of the most sustainable design alternatives. The designers can monitor the economic impact of different design strategies in order to identify the most appropriate option.

I giochi di strategia nell'educazione matematica - il caso del bridge

La tesi evidenzia quanto importante sia l'uso dei giochi di strategia per la didattica della Matematica con particolare attenzione per il gioco del Bridge. Fornisce qualche strumento in più per appassionare lo studente alla matematica. I temi affrontati sono i seguenti: La probabilità con l'uso delle carte, la Matematica che occorre ad un giocatore di bridge per migliorare le proprie prestazioni, i problemi di bridge che offrono stimoli per lo studio della matematica e il Bridge come sport della mente.

Boundary condition identification of a bridge pier using experimental data and FEM modal updating

Over the past twenty years, new technologies have required an increasing use of mathematical models in order to understand better the structural behavior: finite element method is the one mostly used. However, the reliability of this method applied to different situations has to be tried each time. Since it is not possible to completely model the reality, different hypothesis must be done: these are the main problems of FE modeling. The following work deals with this problem and tries to figure out a way to identify some of the unknown main parameters of a structure. This main research focuses on a particular path of study and development, but the same concepts can be applied to other objects of research. The main purpose of this work is the identification of unknown boundary conditions of a bridge pier using the data acquired experimentally with field tests and a FEM modal updating process. This work doesn’t want to be new, neither innovative. A lot of work has been done during the past years on this main problem and many solutions have been shown and published. This thesis just want to rework some of the main aspects of the structural optimization process, using a real structure as fitting model.