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Abstract: Forest environments are exposed to multiple stressful factors of both abiotic and biotic nature which may lead to their massive decline [1]. Understanding the molecular mechanisms of specific stress conditions and monitoring the fluctuations of reliable forest plant biomarkers with affordable methods would be instrumental for assessing stress levels over the time. Ascorbate peroxidase (APX) represents a suitable plant biomarker. APX is a hydrogen peroxide-scavenging enzyme the critical role of which has been described in several plants, both herbaceous and woody. Its activity generally increases under oxidative stress during which its peroxide detoxifying function is part of the wider ascorbate-glutathione cycle [2]. The development of reagents to detect such fluctuations would help the evaluation of plant physiological conditions. In this study, nanobodies (Nbs) targeting APX have been identified. Nbs correspond to the variable domain of heavy chain-only antibodies derived from camelids. They are small (15 kDa), stable, and can be easily produced in bacteria fused to different protein tags according to the downstream applications [3]. After their isolation by biopanning against soluble APX, they have been produced and underwent a biophysical characterization in combination with their antigen (APX-Nb complex) to identify the best binders in terms of stability and affinity. The protein complex characterization was supported by Instruct-ERIC and mainly performed at the BIOCEV institute of Prague. Data from Mass Photometry and Dynamic Light scattering evidenced the formation of the protein complexes, whereas the preliminary data of Hydrogen-Deuterium Exchange Mass Spectrometry, performed with the aim of identifying the residues involved in the paratope/epitope interface, were insufficient to clarify the issue and rather suggested that the interaction has low affinity. This indication was then confirmed by ELISA assay. The combination of multiple methods allowed a comprehensive sample characterization which will require further structural analyses to provide a complete picture of the APX-Nb complex. [1] G. Marie. B. C. M. B. C. Walters, “Forest decline and tree mortality in a southeastern Ohio oak-hickory forest,” Ohio Journal of Science , vol. 97, 1997. [2] O. Chew, J. Whelan, and A. H. Millar, “Molecular Definition of the Ascorbate-Glutathione Cycle in Arabidopsis Mitochondria Reveals Dual Targeting of Antioxidant Defenses in Plants,” Journal of Biological Chemistry, vol. 278, no. 47, 2003, doi: 10.1074/jbc.M307525200. [3] S. Muyldermans, “A guide to: generation and design of nanobodies,” FEBS J, vol. 288, no. 7, pp. 2084–2102, Apr. 2021, doi: 10.1111/febs.15515.
Keywords: nanobody, ascorbate peroxidase, plant stress, protein complex, biophysical methodologies
Published in RUNG: 31.05.2024; Views: 168; Downloads: 0
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Abstract: In these last twenty years a new sensibility for the seismic themes around buildings has emerged from a cultural and technical point of view. As a result of some earthquakes happened in Italy, the promulgation of technical codes has accelerated and, essentially, the standards of Eurocodes were accepted. Consequently, also in the field of historical buildings, a set of specific guidelines have been issued. In the current state of art, designers are faced with many methods of calculation which the technical codes offer for the assessment of the seismic vulnerability of historical buildings. Designers can use simplified or complex approaches which assume different starting hypothesisto obtain different results. This thesis aims to investigate the various aspects that the designer meets during the study of the static behaviour of an historical building and the assessment of its seismic vulnerability. A study case is also proposed which regards a Venetian Villa of the XVIth Century located in “Riviera del Brenta” near Venice, with a critical comparison of the results obtained by the application of the various methods of calculation. The thesis begins with an outline of the historical development of seismic technical codes in Italy and shows how the sensibility has developed in the Italian culture. In particular it is illustrated the difference between the approach of the legislation "a posteriori" (where a “patchy zoning” was generally performed after the occurrence of earthquakes), and the probabilistic approach based on the peaking ground acceleration expected in every Italian site. The Second and the Third Chapters contain the analysis of the current seismic legislations and in particular the guidelines for historical buildings. The Chapter 4 provides a general discussion on the masonry and the definition of the mechanical parameters. There are many critical aspects relating to a characterization of models which can realistically represent the historical masonry. They are due to a heterogeneity of variables such as, for instance, the mechanical parameters, the physiological behaviour, the connections, etc. In the Chapter 5 the basic concepts of seismic behaviour of historical buildings are discussed. In particular the Pagano’s classification of masonry buildings is illustrated with special regard of the following factors: stiffness of horizontal structures; efficiency of “masonry beams”; efficiency of intersection between walls. From the Chapter 6 the analysis of the study case begins. Here we can find the description of the Venetian Villa (historical evolution of the buildings, description of geometrical characteristics, structural behaviour, etc. In the Chapter 7, the first level of analysis is performed for the study-case. The method of calculation is contained in the guidelines for historical buildings. A critical discussion of the results is also illustrated. In the Chapter 8 is performed a linear kinematic analysis based on various possible kinematic mechanisms identified through the study of cracking and the lacks of the structural connections. The Chapter 9 shows a non-linear static analysis (pushover) for the study-case which refers to two limit behaviour models: the first one with infinitely rigid diaphragms and efficient masonry beams; the second one based on infinitely flexible diaphragms and weak masonry beams. The results are compared and critically discussed. In the same way the Chapter 10 and 11 perform a linear static analysis and a linear dynamic analysis for the two models of the Chapter 9, so the results can be compared. In the last Chapter a general discussion about all the results of the performed analysis is conducted. Here the work points out the conceptual limits of each method of calculation and the possibility of use related to the objectives which must be reached. Finally some design indications related to the results of the analysis are illustrated.
Keywords: Seismic, Vulneraility, Analitical, Methodologies
Published in RUNG: 02.07.2020; Views: 2981; Downloads: 42
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