In vitro synthesis of the light-harvesting complex into artificial membrane systems

In green plants, the function of collecting solar energy for photosynthesis is fulfilled by a series of light-harvesting complexes (LHC). The light-harvesting chlorophyll a/b protein (LHCP) is synthesized in the cytosol as a precursor (pLHCP), then imported into chloroplasts and assembled into photosynthetic thylakoid membranes. Knowledge about the regulation of the transport processes of LHCP is rather limited. Closely mimicking the in vivo situation, cell-free protein expression system is employed in this dissertation to study the reconstitution of LHCP into artificial membranes. The approach starts merely from the genetic information of the protein, so the difficult and time-consuming procedures of protein expression and purification can be avoided. The LHCP encoding gene from Pisum sativum was cloned into a cell-free compatible vector system and the protein was expressed in wheat germ extracts. Vesicles or pigment-containing vesicles were prepared with either synthetic lipid or purified plant leaf lipid to mimic cell membranes. LHCP was synthesized in wheat germ extract systems with or without supplemented lipids. The addition of either synthetic or purified plant leaf lipid was found to be beneficial to the general productivity of the expression system. The lipid membrane insertion of the LHCP was investigated by radioactive labelling, protease digestion, and centrifugation assays. The LHCP is partially protected against protease digestion; however the protection is independent from the supplemented lipids.

Controlled reproduction in Anguilla anguilla (L.): advanced studies on broodstock management, spawning techniques and system design for artificial seed production.

The thesis aims to expose the advances achieved in the practices of captive breeding of the European eel (Anguilla anguilla). Aspects investigated concern both approaches livestock (breeding selection, response to hormonal stimulation, reproductive performance, incubation of eggs) and physiological aspects (endocrine plasma profiles of players), as well as engineering aspects. Studies conducted on various populations of wild eel have shown that the main determining factor in the selection of wild females destined to captive breeding must be the Silver Index which may determine the stage of pubertal development. The hormonal induction protocol adopted, with increasing doses of carp pituitary extract, it has proven useful to ovarian development, with a synchronization effect that is positively reflected on egg production. The studies on the effects of photoperiod show how the condition of total darkness can positively influence practices of reproductions in captivity. The effects of photoperiod were also investigated at the physiological level, observing the plasma levels of steroids ( E2, T) and thyroid hormones (T3 and T4) and the expression in the liver of vitellogenin (vtg1 and vtg2) and estradiol membrane receptor (ESR1). From the comparison between spontaneous deposition and insemination techniques through the stripping is inferred as the first ports to a better qualitative and quantitative yield in the production of eggs capable of being fertilized, also the presence of a percentage of oocytes completely transparent can be used to obtain eggs at a good rate of fertility. Finally, the design and implementation of a system for recirculating aquaculture suited to meet the needs of species-specific eel showed how to improve the reproductive results, it would be preferable to adopt low-flow and low density incubation.

Novel porphyrin amino acids as building blocks for artificial photosynthetic reaction centers : photoinduced energy and electron transfer

This thesis reports on the synthesis and characterisation of trans-(M)AB2C meso-substituted porphyrin amino acid esters (PAr) (M = 2H or Zn) with tunable electron donating and electron withdrawing Ar substituents at B positions (Ar = 4-C6H4OnBu, 4-C6H4OMe, 2,4,6-C6H2Me3, 4-C6H4Me, C6H5, 4-C6H4F, 4-C6H4CF3, C6F5). These porphyrins were used as key building blocks for photosynthetic LHC (LHC = light-harvesting antenna complex) and RC (RC = reaction center) model compounds.rnBased on free-base or zinc(II) porphyrin amino acid esters and porphyrin acids several amide linked free-base bis(porphyrins) PAr1-PAr2 (Ar1 = 2,4,6-C6H2Me3, C6F5 and Ar2 = 2,4,6-C6H2Me3, 4-C6H4F, 4-C6H4CF3, C6F5), mono metallated bis(porphyrin) PAr1-(Zn)PAr2 (Ar1 = 2,4,6-C6H2Me3 and Ar2 =4-C6H4F) and its doubly zincated complexes (Zn)PAr1-(Zn)PAr2 were prepared. In the fluorescence spectra of free-base bis(porphyrins) the porphyrin with the strongest electron donating power of Ar substituents at B positions is the light emitting unity. The emission of mono metallated bis(porphyrin) occurs only from the free-base porphyrin building block. This phenomenon is caused by an efficient energy transfer likely via the Dexter through-bond mechanism.rnLinking of anthraquinone (Q) as electron acceptor (A) to the N-terminus of porphyrin amino acid esters ((M)PAr) and aminoferrocene (Fc) as electron donor (D) to the C-terminus of the porphyrin resulting in Q-(M)PAr-Fc triads (M = 2H or Zn, Ar = 4-C6H4OnBu, 4-C6H4OMe, 2,4,6-C6H2Me3, 4-C6H4Me, C6H5, 4-C6H4F, 4-C6H4CF3, C6F5) with tunable electron density at the porphyrin chromophore. In these triads initial oxidative PET (Q←(M)PAr) and reductive PET ((M)PAr→Fc) (PET = photoinduced electron transfer) are possible. Both processes leads to an emission quenching of (M)PAr. The efficiency of the PET pathways occurring in the Marcus normal region is controlled by the specific porphyrin electron density.rnAmide-linked conjugates PAr-Fc (Ar = 2,4,6-C6H2Me3, C6F5) and Fmoc-Fc-PAr1 (N-Fmoc-Fc = N-Fmoc protected 1,1’-ferrocene amino acid; Ar1 = C6H5, 4-C6H4F, 4-C6H4CF3, C6F5) as well as hinges PAr2-Fc-PAr1 (Ar1 = C6H5, 4-C6H4F and Ar2 = 2,4,6-C6H2Me3) were studied with respect to the reductive PET. The PET driving force (−GET) in dyads increases with the increasing electron withdrawing character of Ar substituents. Additionally, intramolecular energy transfer between porphyrins PAr1 and PAr2 is feasible in the hinges via the Förster mechanism.rn

Reproduction and artificial restocking of Acipenser naccarii (Bonaparte, 1836). Genetic and physiological characterization through microsatellite markers and radioimmunoassay of sexual steroids

The Adriatic sturgeon, Acipenser naccarii (Bonaparte, 1836), is a highly threatened species due to human activities, particularly overfishing and habitat destruction. Its peculiar ecology and biology (restricted areal and anadromy) makes this species particularly vulnerable. In March 2010 the IUCN has identified the Adriatic sturgeon as a critically endangered species according to the Red List of Threatened Species. Due to its rapid decline, starting from the 80s, at present there is no evidence of natural reproduction in wild environment, which makes the Adriatic sturgeon dependenton captive breeding programs that need to be improved in order to be effective for the survival of the species. For this purpose this study aims to characterize artificial restocking population of Adriatic sturgeon, with both genetic and physiological analysis in order to establish an efficient restocking program for future reproductions. The research is structured on two levels: First genetically, by analyzing 9 microsatellite loci. This gives information relatively about parent allocation and kinship between individuals that were sampled for this study. Hence to predict which reproduction events are the most optimal in terms of incrementing genetic diversity, by the estimation of multilocus pairwise band sharing coefficients. Second step, physiological analysis: testosterone (T) concentration levels in each individual were measured for sexing, without sacrificing the lives of the animals with the use of an invasive examination of the gonads. The combination of interdisciplinary analysis is important to obtain an overall picture in order to indicate the main broodstock participating in reproduction events and future optimal potential participants, in order to ensure a valid management for restocking program and their monitoring.

Synthesis and characterization of artificial aggregates and their potential applications in road construction

L’obiettivo del progetto è stato quello di realizzare ed analizzare aggregati artificiali creati attraverso geopolimerizzazione e macro-incapsulazione di paraffina in aggregati leggeri espansi, discutendo i loro possibili impieghi nelle pavimentazioni stradali. Dopo un'accurata calibrazione delle miscele geopolimeriche, sono stati realizzati degli aggregati artificiali, in seguito caratterizzati in accordo con la norma UNI EN 10343, con l'intento di sostituire materiali stradali vergini. Contemporaneamente, sono stati prodotti aggregati leggeri impregnati di paraffina (PCM), in grado di cambiare fase una volta raggiunti all'incirca i 3 °C, e successivamente rivestiti da due strati di resina poliestere e polvere di granito, denominati PLA: sfruttandone le proprietà termiche, si è valutato il loro possibile utilizzo come soluzione anti-icing. L’ultima fase della ricerca è stata incentrata nella realizzazione di aggregati geopolimerici espansi e molto porosi che potessero contenere una elevata quantità di PCM, sostituendo l'argilla espansa utilizzata nella produzione degli PLA.

The design of artificial nestboxes for the study of secondary hole-nesting birds: a review of methodological inconsistencies and potential biases

The widespread use of artificial nestboxes has led to significant advances in our knowledge of the ecology, behaviour and physiology of cavity nesting birds, especially small passerines Nestboxes have made it easier to perform routine monitoring and experimental manipulation of eggs or nestlings, and also repeatedly to capture, identify and manipulate the parents However, when comparing results across study sites the use of nestboxes may also Introduce a potentially significant confounding variable in the form of differences in nestbox design amongst studies, such as their physical dimensions, placement height, and the way in which they are constructed and maintained However, the use of nestboxes may also introduce an unconsidered and potentially significant confounding variable clue to differences in nestbox design amongst studies, such as their physical dimensions, placement height, and the way in which they are constructed and maintained Here we review to what extent the characteristics of artificial nestboxes (e g size, shape, construction material, colour) are documented in the 'methods' sections of publications involving hole-nesting passerine birds using natural or excavated cavities or artificial nestboxes for reproduction and roosting Despite explicit previous recommendations that authors describe in detail the characteristics of the nestboxes used, we found that the description of nestbox characteristics in most recent publications remains poor and insufficient We therefore list the types of descriptive data that should be included in the methods sections of relevant manuscripts and justify this by discussing how variation in nestbox characteristics can affect or confound conclusions from nestbox studies We also propose several recommendations to improve the reliability and usefulness of research based on long-term studies of any secondary hole-nesting species using artificial nestboxes for breeding or roosting.

Ideal site for ventricular anchoring of artificial chordae in mitral regurgitation

Surgical treatment of mitral leaflet prolapse using artificial neochordae shows excellent outcomes. Upcoming devices attempt the same treatment in a minimally invasive way but target the left ventricular apex as an anchoring point, rather than the tip of the corresponding papillary muscle. In this study, cine cardiac magnetic resonance imaging was used to compare these 2 different anchoring positions and their dynamic relationship with the mitral leaflets.

Evaluating the Impact of the Number of Access Points in Mobile Robots Localization Using Artificial Neural Networks

Localization is information of fundamental importance to carry out various tasks in the mobile robotic area. The exact degree of precision required in the localization depends on the nature of the task. The GPS provides global position estimation but is restricted to outdoor environments and has an inherent imprecision of a few meters. In indoor spaces, other sensors like lasers and cameras are commonly used for position estimation, but these require landmarks (or maps) in the environment and a fair amount of computation to process complex algorithms. These sensors also have a limited field of vision. Currently, Wireless Networks (WN) are widely available in indoor environments and can allow efficient global localization that requires relatively low computing resources. However, the inherent instability in the wireless signal prevents it from being used for very accurate position estimation. The growth in the number of Access Points (AP) increases the overlap signals areas and this could be a useful means of improving the precision of the localization. In this paper we evaluate the impact of the number of Access Points in mobile nodes localization using Artificial Neural Networks (ANN). We use three to eight APs as a source signal and show how the ANNs learn and generalize the data. Added to this, we evaluate the robustness of the ANNs and evaluate a heuristic to try to decrease the error in the localization. In order to validate our approach several ANNs topologies have been evaluated in experimental tests that were conducted with a mobile node in an indoor space.