Drug of abuse analysis: new methods in herbal and biological matrices for medicinal chemistry and forensic investigation

In this Ph.D. project, original and innovative approaches for the quali-quantitative analysis of abuse substances, as well as therapeutic agents with abuse potential and related compounds were designed, developed and validated for application to different fields such as forensics, clinical and pharmaceutical. All the parameters involved in the developed analytical workflows were properly and accurately optimised, from sample collection to sample pretreatment up to the instrumental analysis. Advanced dried blood microsampling technologies have been developed, able of bringing several advantages to the method as a whole, such as significant reduction of solvent use, feasible storage and transportation conditions and enhancement of analyte stability. At the same time, the use of capillary blood allows to increase subject compliance and overall method applicability by exploiting such innovative technologies. Both biological and non-biological samples involved in this project were subjected to optimised pretreatment techniques developed ad-hoc for each target analyte, making also use of advanced microextraction techniques. Finally, original and advanced instrumental analytical methods have been developed based on high and ultra-high performance liquid chromatography (HPLC,UHPLC) coupled to different detection means (mainly mass spectrometry, but also electrochemical, and spectrophotometric detection for screening purpose), and on attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) for solid-state analysis. Each method has been designed to obtain highly selective, sensitive yet sustainable systems and has been validated according to international guidelines. All the methods developed herein proved to be suitable for the analysis of the compounds under investigation and may be useful tools in medicinal chemistry, pharmaceutical analysis, within clinical studies and forensic investigations.

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.

Synthesis and application of new ion-tagged ligands and organocatalysts

We report the synthesis and application of some ion-tagged catalysts in organometallic catalysis and organocatalysis. With the installation of an ionic group on the backbone of a known catalyst, two main effects are generally obtained. i) a modification of the solubility of the catalyst: if judicious choice of the ion pair is made, the ion-tag can confer to the catalyst a solubility profile suitable for catalyst recycling. ii) the ionic group can play a non-innocent role in the process considered: if stabilizing interaction between the ionic group and the developing charges in the transition state are established, the reaction can speed up. We describe the use of ion-tagged diphenylprolinol as Zn ligand. The chiral ligand grafted onto an ionic liquid (IL) was recycled 10 times with no loss of reactivity and selectivity, when it was employed in the first example of enantioselective addition of ZnEt2 to aldehydes in ILs. An ammonium-tagged phosphine displayed the capability to stabilize Pd catalysts for the Suzuki reaction in ILs. The ionic phase was recycled 6 times with no detectable loss of activity and very low Pd leaching in the organic phase. This catalytic system was also employed for the functionalization of the challenging substrate 5,11-dibromotetracene. In the field of organocatalysis, we prepared two ion-tagged derivatives of the McMillan imidazolidinone. The results of the asymmetric Diels-Alder reaction between trans-cinnamaldehyde and cyclopentadiene exhibited great dependence on the position and nature of the ionic group. Finally, when O-TMS-diphenylprolinol was tagged with an imidazolium ion, exploiting a silyl ether linker, an efficient catalyst for the asymmetric addition of aldehydes to nitroolefins was achieved. The catalyst displayed enhanced reactivity and the same high level of selectivity of the untagged parent catalyst and it could be employed in a wide range of reaction conditions, included use of water as solvent.

The effect of osmolytes on protein fold stability at the single-molecule level

By pulling and releasing the tension on protein homomers with the Atomic Force Miscroscope (AFM) at different pulling speeds, dwell times and dwell distances, the observed force-response of the protein can be fitted with suitable theoretical models. In this respect we developed mathematical procedures and open-source computer codes for driving such experiments and fitting Bell’s model to experimental protein unfolding forces and protein folding frequencies. We applied the above techniques to the study of proteins GB1 (the B1 IgG-binding domain of protein G from Streptococcus) and I27 (a module of human cardiac titin) in aqueous solutions of protecting osmolytes such as dimethyl sulfoxide (DMSO), glycerol and trimethylamine N-oxide (TMAO). In order to get a molecular understanding of the experimental results we developed an Ising-like model for proteins that incorporates the osmophobic nature of their backbone. The model benefits from analytical thermodynamics and kinetics amenable to Monte-Carlo simulation. The prevailing view used to be that small protecting osmolytes bridge the separating beta-strands of proteins with mechanical resistance, presumably shifting the transition state to significantly higher distances that correlate with the molecular size of the osmolyte molecules. Our experiments showed instead that protecting osmolytes slow down protein unfolding and speed-up protein folding at physiological pH without shifting the protein transition state on the mechanical reaction coordinate. Together with the theoretical results of the Ising-model, our results lend support to the osmophobic theory according to which osmolyte stabilisation is a result of the preferential exclusion of the osmolyte molecules from the protein backbone. The results obtained during this thesis work have markedly improved our understanding of the strategy selected by Nature to strengthen protein stability in hostile environments, shifting the focus from hypothetical protein-osmolyte interactions to the more general mechanism based on the osmophobicity of the protein backbone.

L'identita visiva pubblica. Nuovi paradigmi di comunicazione

La ricerca proposta affronta il tema dell’identità visiva — uno dei più caratterizzanti e complessi del progetto di comunicazione — applicato alla dimensione pubblica, partendo dalla comprensione e dalla misurazione del profondo mutamento che ha portato alla sua trasformazione da stemma a marchio e poi a logo, da immagine coordinata a brand a progetto di service e experience. La ricognizione dell’evoluzione della comunicazione pubblica e l’analisi del contesto dell’eredità culturale della “grafica di pubblica utilità” forniscono il primo framework di indagine nel quale si prospetta una lettura della complessità della trasformazione del progetto di identità visiva come occasione per un suo ripensamento. Un secondo framework è individuato nel contesto della comunicazione pubblica italiana in cui l’organizzazione e la progettazione di un sistema “istituzione-design-cittadino” è ancora in transizione. L’analisi dell’evoluzione normativa, dei touchpoint, delle pratiche progettuali, degli strumenti e dei ruoli permette di inquadrare gli attuali spazi e paradigmi di interazione che vedono nel servizio digitale la forma prevalente di relazione tra cittadini e dimensione pubblica Attraverso l'esperienza diretta, l’analisi dei casi studio come strumento di indagine e la sistematizzazione di cinque cluster, la tesi propone una rinnovata definizione di identità visiva pubblica che, distaccandosi da una predominante connotazione marketing-oriented, sia funzionale alla costruzione di esperienze utili, e percezioni di queste, rafforzando e stimolando nuovi paradigmi di relazione tra cittadini e dimensione pubblica. Si propone inoltre una prospettiva progettuale che evolve dalla necessità di individuare nuovi percorsi e modalità di rapportarsi con il progetto di pubblica utilità e per la cittadinanza che rifuggono la manifestazione dell’equivoco - piuttosto comune nel contesto pubblico italiano - del confrontarsi con i cosiddetti «wicked problems» (Buchanan, 1992) ricercando, in termini di comunicazione pubblica soluzioni chiuse ed esclusivamente formali, ritenendo che queste possano avere una reale efficacia nel plasmare i rapporti tra cittadini e dimensione pubblica.

Myanmar higher education in transition: the interplay between state authority, student politics and international actors.

The Myanmar “period of transition” (2011-2021) has often been described as a puzzle. Various scholars have begun to engage with the Myanmar context in an effort to grasp the essence of the transition it underwent during President Thein Sein’s USPD and Aung San Suu Kyi’s NLD governments. My work focuses on a specific policy sector, higher education, with a view to contributing to this scholarly debate regarding what was actually happening inside this complex country “transition”, especially in terms of collective participation in the process of political and social change. Reviewing existing scholarly literature on the politics of higher education, my study employs a triangle of analysis in which higher education reform is framed as the interplay of action on the part of “state authority”, “student politics” and “international actors”. What does this interplay lens reveal if we consider Myanmar’s “period of transition”? I argue that it shows the ambiguity and contradiction of tangible pushes for progressive social change that coexisted with authoritarian currents and the reinforcement of the societal position of dominant elites. At the policy level, ultimately, a convergence of interests between international actors and state authority served as the force driving the new higher education reform towards a neo-liberal model of governance and autonomy. This work unpacks the higher education reform process thanks to qualitative data gathered through extensive participant observation, in-depth interviewing and critical discourse analysis, shedding light on the rich narratives of those involved in the politics of higher education in Myanmar.

Analysis of charge-transport properties in GST materials for next generation phase-change memory devices

The quest for universal memory is driving the rapid development of memories with superior all-round capabilities in non-volatility, high speed, high endurance and low power. The memory subsystem accounts for a significant cost and power budget of a computer system. Current DRAM-based main memory systems are starting to hit the power and cost limit. To resolve this issue the industry is improving existing technologies such as Flash and exploring new ones. Among those new technologies is the Phase Change Memory (PCM), which overcomes some of the shortcomings of the Flash such as durability and scalability. This alternative non-volatile memory technology, which uses resistance contrast in phase-change materials, offers more density relative to DRAM, and can help to increase main memory capacity of future systems while remaining within the cost and power constraints. Chalcogenide materials can suitably be exploited for manufacturing phase-change memory devices. Charge transport in amorphous chalcogenide-GST used for memory devices is modeled using two contributions: hopping of trapped electrons and motion of band electrons in extended states. Crystalline GST exhibits an almost Ohmic I(V) curve. In contrast amorphous GST shows a high resistance at low biases while, above a threshold voltage, a transition takes place from a highly resistive to a conductive state, characterized by a negative differential-resistance behavior. A clear and complete understanding of the threshold behavior of the amorphous phase is fundamental for exploiting such materials in the fabrication of innovative nonvolatile memories. The type of feedback that produces the snapback phenomenon is described as a filamentation in energy that is controlled by electron–electron interactions between trapped electrons and band electrons. The model thus derived is implemented within a state-of-the-art simulator. An analytical version of the model is also derived and is useful for discussing the snapback behavior and the scaling properties of the device.

Inverse Static Analysis of Massive Parallel Arrays of Three-State Actuators via Artificial Intelligence

Massive parallel robots (MPRs) driven by discrete actuators are force regulated robots that undergo continuous motions despite being commanded through a finite number of states only. Designing a real-time control of such systems requires fast and efficient methods for solving their inverse static analysis (ISA), which is a challenging problem and the subject of this thesis. In particular, five Artificial intelligence methods are proposed to investigate the on-line computation and the generalization error of ISA problem of a class of MPRs featuring three-state force actuators and one degree of revolute motion.

Unsupervised reinforcement learning via state entropy maximization

Reinforcement Learning (RL) provides a powerful framework to address sequential decision-making problems in which the transition dynamics is unknown or too complex to be represented. The RL approach is based on speculating what is the best decision to make given sample estimates obtained from previous interactions, a recipe that led to several breakthroughs in various domains, ranging from game playing to robotics. Despite their success, current RL methods hardly generalize from one task to another, and achieving the kind of generalization obtained through unsupervised pre-training in non-sequential problems seems unthinkable. Unsupervised RL has recently emerged as a way to improve generalization of RL methods. Just as its non-sequential counterpart, the unsupervised RL framework comprises two phases: An unsupervised pre-training phase, in which the agent interacts with the environment without external feedback, and a supervised fine-tuning phase, in which the agent aims to efficiently solve a task in the same environment by exploiting the knowledge acquired during pre-training. In this thesis, we study unsupervised RL via state entropy maximization, in which the agent makes use of the unsupervised interactions to pre-train a policy that maximizes the entropy of its induced state distribution. First, we provide a theoretical characterization of the learning problem by considering a convex RL formulation that subsumes state entropy maximization. Our analysis shows that maximizing the state entropy in finite trials is inherently harder than RL. Then, we study the state entropy maximization problem from an optimization perspective. Especially, we show that the primal formulation of the corresponding optimization problem can be (approximately) addressed through tractable linear programs. Finally, we provide the first practical methodologies for state entropy maximization in complex domains, both when the pre-training takes place in a single environment as well as multiple environments.