Design and synthesis of new (E)-cinnamic N-acylhydrazones as potent antitrypanosomal agents

We report herein the synthesis and trypanocidal profile of new (E)-cinnamic N-acylhydrazones (NAHs) designed by exploiting molecular hybridization between the potent cruzain inhibitors (E)-1-(benzo[d] 11,3)dioxol-5-yl)-3-(4-bromophenyl)prop-2-en-1-one and (E)-3-hydroxy-N'-((2-hydroxynaphthalen-1-yl)methylene)-7-methoxy-2-naphthohydrazide. These derivatives were evaluated against both amastigote and trypomastigote forms of Trypanosoma cruzi and lead us to identify two compounds that were approximately two times more active than the reference drug, benznidazole, and with good cytotoxic index. Although designed as cruzain inhibitors, the weak potency displayed by the best cinnamyl NAH derivatives indicated that another mechanism of action was likely responsible for their trypanocide action. (C) 2012 Elsevier Masson SAS. All rights reserved.

Design and development of new pressure sensors for aerodynamic applications

This artwork reports on two different projects that were carried out during the three years of Doctor of the Philosophy course. In the first years a project regarding Capacitive Pressure Sensors Array for Aerodynamic Applications was developed in the Applied Aerodynamic research team of the Second Faculty of Engineering, University of Bologna, Forlì, Italy, and in collaboration with the ARCES laboratories of the same university. Capacitive pressure sensors were designed and fabricated, investigating theoretically and experimentally the sensor’s mechanical and electrical behaviours by means of finite elements method simulations and by means of wind tunnel tests. During the design phase, the sensor figures of merit are considered and evaluated for specific aerodynamic applications. The aim of this work is the production of low cost MEMS-alternative devices suitable for a sensor network to be implemented in air data system. The last two year was dedicated to a project regarding Wireless Pressure Sensor Network for Nautical Applications. Aim of the developed sensor network is to sense the weak pressure field acting on the sail plan of a full batten sail by means of instrumented battens, providing a real time differential pressure map over the entire sail surface. The wireless sensor network and the sensing unit were designed, fabricated and tested in the faculty laboratories. A static non-linear coupled mechanical-electrostatic simulation, has been developed to predict the pressure versus capacitance static characteristic suitable for the transduction process and to tune the geometry of the transducer to reach the required resolution, sensitivity and time response in the appropriate full scale pressure input A time dependent viscoelastic error model has been inferred and developed by means of experimental data in order to model, predict and reduce the inaccuracy bound due to the viscolelastic phenomena affecting the Mylar® polyester film used for the sensor diaphragm. The development of the two above mentioned subjects are strictly related but presently separately in this artwork.

Heave of large excavations in clay: modelling and analysis of two british case-histories

ABSTRACT Il presente lavoro vuole introdurre la problematica del rigonfiamento del terreno a seguito di grandi scavi in argilla. Il sollevamento del terreno dopo lo scavo può passare inosservato ma sono numerosi i casi in cui il rigonfiamento dura per molti anni e addirittura decenni, Shell Centre, London, Lion Yard, Cambridge, Bell Common, London, ecc. Questo rigonfiamento il più delle volte è impedito dalla presenza di fondazioni, si genera quindi una pressione distribuita che se non considerata in fase di progetto può portare alla fessurazione della fondazione stessa. L’anima del progetto è la modellazione e l’analisi del rigonfiamento di grandi scavi in argilla, confrontando poi i risultati con i dati reali disponibili in letteratura. L’idea del progetto nasce dalla difficoltà di ottenere stime e previsioni attendibili del rigonfiamento a seguito di grandi scavi in argilla sovraconsolidata. Inizialmente ho esaminato la teoria e i fattori che influenzano il grado e la velocità del rigonfiamento, quali la rigidezza, permeabilità, fessurazione, struttura del suolo, etc. In seguito ho affrontato lo studio del comportamento rigonfiante di argille sovraconsolidate a seguito di scarico tensionale (scavi), si è evidenziata l’importanza di differenziare il rigonfiamento primario e il rigonfiamento secondario dovuto al fenomeno del creep. Il tema centrale del progetto è l’analisi numerica tramite Flac di due grandi scavi in argilla, Lion Yard, Cambridge, e, Bell Common, London. Attraverso una dettagliata analisi parametrica sono riuscito a trovare i migliori parametri che modellano il comportamento reale nei due casi in esame, in questo modo è possibile arrivare a stime e previsioni attendibili del fenomeno rigonfiante del terreno a seguito di grandi scavi. Gli scavi modellati Lion Yard e Bell Common sono rispettivamente in Gault Clay e London Clay, grazie a famosi recenti articoli scientifici sono riuscito a evidenziare la principali propietà che diversificano i due terreni in esame, tali propietà sono estremamente differenti dalle normali caratteristiche considerate per la progettazione in presenza di terreno argilloso; sono così riuscito a implementare i migliori parametri per descrivere il comportamento dei due terreni nei diversi modelli. Ho inoltre studiato l’interazione terreno-struttura, la pressione esercitata dal rigonfiamento del terreno è strettamente funzione delle caratteristiche di connesione tra fondazione superficiale e muro di sostegno, tale pressione non deve essere ignorata in fase progettuale poichè può raggiungere importanti valori. Nello scavo di Lion Yard, considerando la presenza delle fondazioni profonde ho evidenziato il fatto che il rigonfiamento crea una forza distribuita di taglio tra i pali di fondazione ed il terreno, anche tale sollecitazione dovrebbe essere considerata ai fini della progettazione. La problematica non si ferma solo sull’interazione terreno-fondazioni, infatti durante gli scavi di importanti fondazioni londinesi lo scarico tensionale ha creato uno spostamento significativo positivo verso la superfice di tratti di tunnel della metropolita, questo fenomeno può creare seri problemi di sicurezza nella rete dei trasporti pubblici. Infine sono stati messi a confronto i risultati del programma Flac con quelli di metodi semplificati, ho trovato che utilizzando il metodo iterativo di O’Brien i risultati sono simili alla realtà e il tempo di calcolo è molto inferiore di quello richiesto utilizzando Flac, 2-3 giorni. In conclusione posso affermare che grazie ad una dettagliata analisi parametrica è stato possibile stimare il rigonfiamento del terreno, argilla sovraconsolidata, nei due casi analizzati.

Design and Computation of Warped Time-Frequency Transforms

In this work we introduce an analytical approach for the frequency warping transform. Criteria for the design of operators based on arbitrary warping maps are provided and an algorithm carrying out a fast computation is defined. Such operators can be used to shape the tiling of time-frequency plane in a flexible way. Moreover, they are designed to be inverted by the application of their adjoint operator. According to the proposed mathematical model, the frequency warping transform is computed by considering two additive operators: the first one represents its nonuniform Fourier transform approximation and the second one suppresses aliasing. The first operator is known to be analytically characterized and fast computable by various interpolation approaches. A factorization of the second operator is found for arbitrary shaped non-smooth warping maps. By properly truncating the operators involved in the factorization, the computation turns out to be fast without compromising accuracy.

Methodology and analysis of gas embolism: experimental models and stranded cetaceans

Programa de doctorado: Sanidad animal

Design and Characterization of New Luminescent Sensors and Labels

The aim of this Ph.D. project has been the design and characterization of new and more efficient luminescent tools, in particular sensors and labels, for analytical chemistry, medical diagnostics and imaging. Actually both the increasing temporal and spatial resolutions that are demanded by those branches, coupled to a sensitivity that is required to reach the single molecule resolution, can be provided by the wide range of techniques based on luminescence spectroscopy. As far as the development of new chemical sensors is concerned, as chemists we were interested in the preparation of new, efficient, sensing materials. In this context, we kept developing new molecular chemosensors, by exploiting the supramolecular approach, for different classes of analytes. In particular we studied a family of luminescent tetrapodal-hosts based on aminopyridinium units with pyrenyl groups for the detection of anions. These systems exhibited noticeable changes in the photophysical properties, depending on the nature of the anion; in particular, addition of chloride resulted in a conformational change, giving an initial increase in excimeric emission. A good selectivity for dicarboxylic acid was also found. In the search for higher sensitivities, we moved our attention also to systems able to perform amplification effects. In this context we described the metal ion binding properties of three photoactive poly-(arylene ethynylene) co-polymers with different complexing units and we highlighted, for one of them, a ten-fold amplification of the response in case of addition of Zn2+, Cu2+ and Hg2+ ions. In addition, we were able to demonstrate the formation of complexes with Yb3+ an Er3+ and an efficient sensitization of their typical metal centered NIR emission upon excitation of the polymer structure, this feature being of particular interest for their possible applications in optical imaging and in optical amplification for telecommunication purposes. An amplification effect was also observed during this research in silica nanoparticles derivatized with a suitable zinc probe. In this case we were able to prove, for the first time, that nanoparticles can work as “off-on” chemosensors with signal amplification. Fluorescent silica nanoparticles can be thus seen as innovative multicomponent systems in which the organization of photophysically active units gives rise to fruitful collective effects. These precious effects can be exploited for biological imaging, medical diagnostic and therapeutics, as evidenced also by some results reported in this thesis. In particular, the observed amplification effect has been obtained thanks to a suitable organization of molecular probe units onto the surface of the nanoparticles. In the effort of reaching a deeper inside in the mechanisms which lead to the final amplification effects, we also attempted to find a correlation between the synthetic route and the final organization of the active molecules in the silica network, and thus with those mutual interactions between one another which result in the emerging, collective behavior, responsible for the desired signal amplification. In this context, we firstly investigated the process of formation of silica nanoparticles doped with pyrene derivative and we showed that the dyes are not uniformly dispersed inside the silica matrix; thus, core-shell structures can be formed spontaneously in a one step synthesis. Moreover, as far as the design of new labels is concerned, we reported a new synthetic approach to obtain a class of robust, biocompatible silica core-shell nanoparticles able to show a long-term stability. Taking advantage of this new approach we also showed the synthesis and photophysical properties of core-shell NIR absorbing and emitting materials that proved to be very valuable for in-vivo imaging. In general, the dye doped silica nanoparticles prepared in the framework of this project can conjugate unique properties, such as a very high brightness, due to the possibility to include many fluorophores per nanoparticle, high stability, because of the shielding effect of the silica matrix, and, to date, no toxicity, with a simple and low-cost preparation. All these features make these nanostructures suitable to reach the low detection limits that are nowadays required for effective clinical and environmental applications, fulfilling in this way the initial expectations of this research project.

Design and synthesis of novel non peptidomimtic beta-secretase inhibitors in the treatment of Alzheimer's disease

The aspartic protease BACE1 (β-amyloid precursor protein cleaving enzyme, β-secretase) is recognized as one of the most promising targets in the treatment of Alzheimer's disease (AD). The accumulation of β-amyloid peptide (Aβ) in the brain is a major factor in the pathogenesis of AD. Aβ is formed by initial cleavage of β-amyloid precursor protein (APP) by β-secretase, therefore BACE1 inhibition represents one of the therapeutic approaches to control progression of AD, by preventing the abnormal generation of Aβ. For this reason, in the last decade, many research efforts have focused at the identification of new BACE1 inhibitors as drug candidates. Generally, BACE1 inhibitors are grouped into two families: substrate-based inhibitors, designed as peptidomimetic inhibitors, and non-peptidomimetic ones. The research on non-peptidomimetic small molecules BACE1 inhibitors remains the most interesting approach, since these compounds hold an improved bioavailability after systemic administration, due to a good blood-brain barrier permeability in comparison to peptidomimetic inhibitors. Very recently, our research group discovered a new promising lead compound for the treatment of AD, named lipocrine, a hybrid derivative between lipoic acid and the AChE inhibitor (AChEI) tacrine, characterized by a tetrahydroacridinic moiety. Lipocrine is one of the first compounds able to inhibit the catalytic activity of AChE and AChE-induced amyloid-β aggregation and to protect against reactive oxygen species. Due to this interesting profile, lipocrine was also evaluated for BACE1 inhibitory activity, resulting in a potent lead compound for BACE1 inhibition. Starting from this interesting profile, a series of tetrahydroacridine analogues were synthesised varying the chain length between the two fragments. Moreover, following the approach of combining in a single molecule two different pharmacophores, we designed and synthesised different compounds bearing the moieties of known AChEIs (rivastigmine and caproctamine) coupled with lipoic acid, since it was shown that dithiolane group is an important structural feature of lipocrine for the optimal inhibition of BACE1. All the tetrahydroacridines, rivastigmine and caproctamine-based compounds, were evaluated for BACE1 inhibitory activity in a FRET (fluorescence resonance energy transfer) enzymatic assay (test A). With the aim to enhancing the biological activity of the lead compound, we applied the molecular simplification approach to design and synthesize novel heterocyclic compounds related to lipocrine, in which the tetrahydroacridine moiety was replaced by 4-amino-quinoline or 4-amino-quinazoline rings. All the synthesized compounds were also evaluated in a modified FRET enzymatic assay (test B), changing the fluorescent substrate for enzymatic BACE1 cleavage. This test method guided deep structure-activity relationships for BACE1 inhibition on the most promising quinazoline-based derivatives. By varying the substituent on the 2-position of the quinazoline ring and by replacing the lipoic acid residue in lateral chain with different moieties (i.e. trans-ferulic acid, a known antioxidant molecule), a series of quinazoline derivatives were obtained. In order to confirm inhibitory activity of the most active compounds, they were evaluated with a third FRET assay (test C) which, surprisingly, did not confirm the previous good activity profiles. An evaluation study of kinetic parameters of the three assays revealed that method C is endowed with the best specificity and enzymatic efficiency. Biological evaluation of the modified 2,4-diamino-quinazoline derivatives measured through the method C, allow to obtain a new lead compound bearing the trans-ferulic acid residue coupled to 2,4-diamino-quinazoline core endowed with a good BACE1 inhibitory activity (IC50 = 0.8 mM). We reported on the variability of the results in the three different FRET assays that are known to have some disadvantages in term of interference rates that are strongly dependent on compound properties. The observed results variability could be also ascribed to different enzyme origin, varied substrate and different fluorescent groups. The inhibitors should be tested on a parallel screening in order to have a more reliable data prior to be tested into cellular assay. With this aim, preliminary cellular BACE1 inhibition assay carried out on lipocrine confirmed a good cellular activity profile (EC50 = 3.7 mM) strengthening the idea to find a small molecule non-peptidomimetic compound as BACE1 inhibitor. In conclusion, the present study allowed to identify a new lead compound endowed with BACE1 inhibitory activity in submicromolar range. Further lead optimization to the obtained derivative is needed in order to obtain a more potent and a selective BACE1 inhibitor based on 2,4-diamino-quinazoline scaffold. A side project related to the synthesis of novel enzymatic inhibitors of BACE1 in order to explore the pseudopeptidic transition-state isosteres chemistry was carried out during research stage at Università de Montrèal (Canada) in Hanessian's group. The aim of this work has been the synthesis of the δ-aminocyclohexane carboxylic acid motif with stereochemically defined substitution to incorporating such a constrained core in potential BACE1 inhibitors. This fragment, endowed with reduced peptidic character, is not known in the context of peptidomimetic design. In particular, we envisioned an alternative route based on an organocatalytic asymmetric conjugate addition of nitroalkanes to cyclohexenone in presence of D-proline and trans-2,5-dimethylpiperazine. The enantioenriched obtained 3-(α-nitroalkyl)-cyclohexanones were further functionalized to give the corresponding δ-nitroalkyl cyclohexane carboxylic acids. These intermediates were elaborated to the target structures 3-(α-aminoalkyl)-1-cyclohexane carboxylic acids in a new readily accessible way.

Reconstruction and analysis of the NF-kB pathway interactome: a systems biology approach

Background. One of the phenomena observed in human aging is the progressive increase of a systemic inflammatory state, a condition referred to as “inflammaging”, negatively correlated with longevity. A prominent mediator of inflammation is the transcription factor NF-kB, that acts as key transcriptional regulator of many genes coding for pro-inflammatory cytokines. Many different signaling pathways activated by very diverse stimuli converge on NF-kB, resulting in a regulatory network characterized by high complexity. NF-kB signaling has been proposed to be responsible of inflammaging. Scope of this analysis is to provide a wider, systemic picture of such intricate signaling and interaction network: the NF-kB pathway interactome. Methods. The study has been carried out following a workflow for gathering information from literature as well as from several pathway and protein interactions databases, and for integrating and analyzing existing data and the relative reconstructed representations by using the available computational tools. Strong manual intervention has been necessarily used to integrate data from multiple sources into mathematically analyzable networks. The reconstruction of the NF-kB interactome pursued with this approach provides a starting point for a general view of the architecture and for a deeper analysis and understanding of this complex regulatory system. Results. A “core” and a “wider” NF-kB pathway interactome, consisting of 140 and 3146 proteins respectively, were reconstructed and analyzed through a mathematical, graph-theoretical approach. Among other interesting features, the topological characterization of the interactomes shows that a relevant number of interacting proteins are in turn products of genes that are controlled and regulated in their expression exactly by NF-kB transcription factors. These “feedback loops”, not always well-known, deserve deeper investigation since they may have a role in tuning the response and the output consequent to NF-kB pathway initiation, in regulating the intensity of the response, or its homeostasis and balance in order to make the functioning of such critical system more robust and reliable. This integrated view allows to shed light on the functional structure and on some of the crucial nodes of thet NF-kB transcription factors interactome. Conclusion. Framing structure and dynamics of the NF-kB interactome into a wider, systemic picture would be a significant step toward a better understanding of how NF-kB globally regulates diverse gene programs and phenotypes. This study represents a step towards a more complete and integrated view of the NF-kB signaling system.

Comparative genomic sequencing analysis of a region in human chromosome 11p15.3, mouse chromosome 7 and analysis of the novel STK33, Stk33 gene

The comparative genomic sequence analysis of a region in human chromosome 11p15.3 and its homologous segment in mouse chromosome 7 between ST5 and LMO1 genes has been performed. 158,201 bases were sequenced in the mouse and compared with the syntenic region in human, partially available in the public databases. The analysed region exhibits the typical eukaryotic genomic structure and compared with the close neighbouring regions, strikingly reflexes the mosaic pattern distribution of (G+C) and repeats content despites its relative short size. Within this region the novel gene STK33 was discovered (Stk33 in the mouse), that codes for a serine/threonine kinase. The finding of this gene constitutes an excellent example of the strength of the comparative sequencing approach. Poor gene-predictions in the mouse genomic sequence were corrected and improved by the comparison with the unordered data from the human genomic sequence publicly available. Phylogenetical analysis suggests that STK33 belongs to the calcium/calmodulin-dependent protein kinases group and seems to be a novelty in the chordate lineage. The gene, as a whole, seems to evolve under purifying selection whereas some regions appear to be under strong positive selection. Both human and mouse versions of serine/threonine kinase 33, consists of seventeen exons highly conserved in the coding regions, particularly in those coding for the core protein kinase domain. Also the exon/intron structure in the coding regions of the gene is conserved between human and mouse. The existence and functionality of the gene is supported by the presence of entries in the EST databases and was in vivo fully confirmed by isolating specific transcripts from human uterus total RNA and from several mouse tissues. Strong evidence for alternative splicing was found, which may result in tissue-specific starting points of transcription and in some extent, different protein N-termini. RT-PCR and hybridisation experiments suggest that STK33/Stk33 is differentially expressed in a few tissues and in relative low levels. STK33 has been shown to be reproducibly down-regulated in tumor tissues, particularly in ovarian tumors. RNA in-situ hybridisation experiments using mouse Stk33-specific probes showed expression in dividing cells from lung and germinal epithelium and possibly also in macrophages from kidney and lungs. Preliminary experimentation with antibodies designed in this work, performed in parallel to the preparation of this manuscript, seems to confirm this expression pattern. The fact that the chromosomal region 11p15 in which STK33 is located may be associated with several human diseases including tumor development, suggest further investigation is necessary to establish the role of STK33 in human health.