Neurophysiological, molecular and pathophysiological aspects of synthetic torpor

Synthetic torpor is a peculiar physiological condition resembling natural torpor, in which even non-hibernating species can be induced through different pharmacological approaches. The growing interest in the induction of a safe synthetic torpor state in non-hibernating species stems from the possible applications that it may have in a translational perspective. In particular, the deeper understanding of the functional changes occurring during and after synthetic torpor may lead to the standardization of a safe procedure to be used also in humans and to the implementation of new therapeutic strategies. Some of the most interesting and peculiar characteristics of torpor that should be assessed in synthetic torpor and may have a translational relevance are: the reversible hyperphosphorylation of neuronal Tau protein, the strong and extended neural plasticity, which may be related to Tau regulatory processes, and the development of radioresistance. In this respect, in the present thesis, rats were induced into synthetic torpor by the pharmacological inhibition of the raphe pallidus, a key brainstem thermoregulatory area, in order to assess: i) whether a reversible hyperphosphorylation of Tau protein occurs at the spinal cord level, also testing the possible involvement of microglia activation in this phenomenon; ii) sleep quality after synthetic torpor and its possible involvement in the process of Tau dephosphorylation; iii) whether synthetic torpor has radioprotective properties, by assessing histopathological and molecular features in animals exposed to X-rays irradiation. The results showed that: i) a reversible hyper-phosphorylation of Tau protein also occurs in synthetic torpor in the dorsal horns of the spinal cord; ii) sleep regulation after synthetic torpor seems to be physiological, and sleep deprivation speeds up Tau dephosphorylation; iii) synthetic torpor induces a consistent increase in radioresistance, as shown by analyses at both histological and molecular level.

Biological analysis of the in-vitro effects of homologous recombination inhibition and its use in cancer treatment through synthetic lethality

Synthetic lethality represents an anticancer strategy that targets tumor specific gene defects. One of the most studied application is the use of PARP inhibitors (e.g. olaparib) in BRCA1/2-less cancer cells. In BRCA2-defective tumors, olaparib (OLA) inhibits DNA single-strand break repair, while BRCA2 mutations hamper homologous recombination (HR) repair. The simultaneous impairment of those pathways leads BRCA-less cells to death by synthetic lethality. The projects described in this thesis were aimed at extending the use of OLA in cancer cells that do not carry a mutation in BRCA2 by combining this drug with compounds that could mimic a BRCA-less environment via HR inhibition. We demonstrated the effectiveness of our “fully small-molecule induced synthetic lethality” by using two different approaches. In the direct approach (Project A), we identified a series of neo-synthesized compounds (named RAD51-BRCA2 disruptors) that mimic BRCA2 mutations by disrupting the RAD51-BRCA2 interaction and thus the HR pathway. Compound ARN 24089 inhibited HR in human pancreatic adenocarcinoma cell line and triggered synthetic lethality by synergizing with OLA. Interestingly, the observed synthetic lethality was triggered by tackling two biochemically different mechanisms: enzyme inhibition (PARP) and protein-protein disruption (RAD51-BRCA2). In the indirect approach (Project B), we inhibited HR by interfering with the cellular metabolism through inhibition of LDH activity. The obtained data suggest an LDH-mediated control on HR that can be exerted by regulating either the energy supply needed to this repair mechanism or the expression level of genes involved in DNA repair. LDH inhibition also succeeded in increasing the efficiency of OLA in BRCA-proficient cell lines. Although preliminary, these results highlight a complex relationship between metabolic reactions and the control of DNA integrity. Both the described projects proved that our “fully small-molecule-induced synthetic lethality” approach could be an innovative approach to unmet oncological needs.

Pastoral Livelihoods in South Ethiopia - Value Chain Assessment of Gum & Resins in Moyale Area

This research assessed the value chain of gum and resins, which are available in four woreda in the southern lowlands of Ethiopia. They are Moyale Somali, Moyale Oromia, Dhas and Dire woreda. The output of this research is the elaboration of three value chains. The first is a general one for all the woreda, while the other two concern the Moyale and Dubluk markets. The assessed products are the gum arabic from Acacia trees and the resin exuded by the dunkhal tree - Boswellia family. The aim of this study was not only to understand the way in which resins and gum gain value through the chain and the profit each stakeholder gains, but more importantly how pastoralists use resin and gum collection to diversify their income. The first chapter analyses what it means to be a pastoralist in the Moyale area and its challenges. The second chapter describes how the policies of the central state influenced the pastoral access to rangelands and water and the way in which this contributed to the increase of conflict among the different groups. A particular focus is on the settlement. The third chapter describes the different ethnic groups living in the studied area and their management system to preserve resources and cope with the dry season. This chapter considers the dynamic evolution of the relations among the various groups in terms of negotiating access to resources while facing political and climatic challenges. The fourth chapter illustrates the physical context and the environment, and the way in which it has been managed in order to preserve the pastoral lifestyle. The fifth chapter describes the characteristics of gum and resins in the studied area. Finally, the sixth chapter describes how the value chain methodology was applied in this specific study and its outputs.

Tailoring, synthesis and structure-property relationships of 2,3-thienoimide based molecular materials

Organic molecular semiconductors are subject of intense research for their crucial role as key components of new generation low cost, flexible, and large area electronic devices such as displays, thin-film transistors, solar cells, sensors and logic circuits. In particular, small molecular thienoimide (TI) based materials are emerging as novel multifunctional materials combining a good processability together to ambipolar or n-type charge transport and electroluminescence at the solid state, thus enabling the fabrication of integrated devices like organic field effect transistors (OFETs) and light emitting transistor (OLETs). Given this peculiar combination of characteristics, they also constitute the ideal substrates for fundamental studies on the structure-property relationships in multifunctional molecular systems. In this scenario, this thesis work is focused on the synthesis of new thienoimide based materials with tunable optical, packing, morphology, charge transport and electroluminescence properties by following a fine molecular tailoring, thus optimizing their performances in device as well as investigating and enabling new applications. Investigation on their structure-property relationships has been carried out and in particular, the effect of different π-conjugated cores (heterocycles, length) and alkyl end chain (shape, length) changes have been studied, obtaining materials with enhanced electron transport capability end electroluminescence suitable for the realization of OFETs and single layer OLETs. Moreover, control on the polymorphic behaviour characterizing thienoimide materials has been reached by synthetic and post-synthetic methodologies, developing multifunctional materials from a single polymorphic compound. Finally, with the aim of synthesizing highly pure materials, simplifying the purification steps and avoiding organometallic residues, procedures based on direct arylation reactions replacing conventional cross-couplings have been investigated and applied to different classes of molecules, bearing thienoimidic core or ends, as well as thiophene and anthracene derivatives, validating this approach as a clean alternative for the synthesis of several molecular materials.

From core rigidities to dynamic capabilities: the role of external knowledge. A multiple case study

This study focuses on the processes of change that firms undertake to overcome conditions of organizational rigidity and develop new dynamic capabilities, thanks to the contribution of external knowledge. When external contingencies highlight firms’ core rigidities, external actors can intervene in change projects, providing new competences to firms’ managers. Knowledge transfer and organizational learning processes can lead to the development of new dynamic capabilities. Existing literature does not completely explain how these processes develop and how external knowledge providers, as management consultants, influence them. Dynamic capabilities literature has become very rich in the last years; however, the models that explain how dynamic capabilities evolve are not particularly investigated. Adopting a qualitative approach, this research proposes four relevant case studies in which external actors introduce new knowledge within organizations, activating processes of change. Each case study consists of a management consulting project. Data are collected through in-depth interviews with consultants and managers. A large amount of documents supports evidences from interviews. A narrative approach is adopted to account for change processes and a synthetic approach is proposed to compare case studies along relevant dimensions. This study presents a model of capabilities evolution, supported by empirical evidence, to explain how external knowledge intervenes in capabilities evolution processes: first, external actors solve gaps between environmental demands and firms’ capabilities, changing organizational structures and routines; second, a knowledge transfer between consultants and managers leads to the creation of new ordinary capabilities; third, managers can develop new dynamic capabilities through a deliberate learning process that internalizes new tacit knowledge from consultants. After the end of the consulting project, two elements can influence the deliberate learning process: new external contingencies and changes in the perceptions about external actors.

Molecole bioattive naturali e sintetiche per la terapia di malattie da aumentato riassorbimento osseo

Le terapie convenzionali per le malattie da aumentato riassorbimento osseo sono limitate dalla tossicità sistemica, bassa biodisponibilità farmacologica e scarsa aderenza alle terapie. In questo studio sono stati considerati approcci terapeutici innovativi basati su composti naturali e sintetici. I) Valutazione dell'attività biologica di composti naturali. Evidenze sperimentali hanno dimostrato l’attività antiproliferativa ed antiapoptotica di piante della Medicina ayurvedica. Queste proprietà sono sfruttabili nel trattamento di malattie da aumentato riassorbimento osseo, come l'osteoporosi. Per chiarire i possibili effetti terapeutici di questi composti, sono stati studiati i decotti di Rubia cordifolia, Hemidesmus indicus, Emblica officinalis, ed Asparagus racemosus. Hemidesmis indicus si è dimostrato il più efficace. II) Valutazione dell'attività biologica di composti sintetici. I bisfosfonati (BP) sono farmaci capaci di legarsi alle superfici minerali ossee e all’idrossiapatite, nei siti di rimodellamento osseo. Poiché i BP inibiscono la funzione degli osteoclasti, sono convenzionalmente impiegati nel trattamento di malattie da aumentato riassorbimento osseo, come l'osteoporosi. Tuttavia, gli elevati costi e gli effetti collaterali legati alla somministrazione determinano una scarsa aderenza al trattamento condizionandone l’efficacia. Scopo di questo studio è stato quello di valutare l'attività biologica di BP chimicamente innovativi, meno tossici e sintetizzati con strategie catalitiche semplificate ed ecocompatibili, in modo da ridurre i costi di produzione. È stato valutato l’effetto citotossico e antiosteoclastico dei composti e confrontato con quello dei BP comunemente impiegati in clinica (neridronato, pamidronato e alendronato). I risultati sono stati considerati raggiunti qualora fossero identificati BP di nuova sintesi non citotossici e capaci di conservare almeno il 90% della capacità dei substrati di base di inibire il riassorbimento osseo. Tutti i composti di nuova sintesi sono risultati meno tossici del BP convenzionale, anche a concentrazioni più elevate ed i più efficaci sono stati un BP coniugato con acido biliare, un BP aromatico contenente azoto ed un BP alifatico contenente zolfo.

3D bioprinted organ models for drug screening

In recent years, 3D bioprinting has emerged as an innovative and versatile technology able to produce in vitro models that resemble the native spatial organization of organ tissues, by employing or more bioinks composed of various types of cells suspended in hydrogels. Natural and semi-synthetic hydrogels are extensively used for 3D bioprinting models since they can mimic the natural composition of the tissues, they are biocompatible and bioactive with customizable mechanical properties, allowing to support cell growth. The possibility to tailor hydrogels mechanical properties by modifying the chemical structures to obtain photo-crosslinkable materials, while maintaining their biocompatibility and biomimicry, make their use versatile and suitable to simulate a broad spectrum of physiological features. In this PhD Thesis, 3D bioprinted in vitro models with tailored mechanical properties and physiologically-like features were fabricated. AlgMa-based bioinks were employed to produce a living platform with gradient stiffness, with the aim to create an easy to handle and accessible biological tool to evaluate mechanobiology. In addition, GelMa, collagen and IPN of GelMa and collagen were used as bioinks to fabricate a proof-of-concept of 3D intestinal barrier, which include multiple cell components and multi-layered structure. A useful rheological guide to drive users to the selection of the suitable bioinks for 3D bioprinting and to correlate the model’s mechanical stability after crosslinking is proposed. In conclusion, a platform capable to reproduce models with physiological gradient stiffness was developed and the fabrication of 3D bioprinted intestinal models displaying a good hierarchical structure and cells composition was fully reported and successfully achieved. The good biological results obtained demonstrated that 3D bioprinting can be used for the fabrications of 3D models and that the mechanical properties of the external environment plays a key role on the cell pathways, viability and morphology.

Synthetic and mechanistic investigation of new radical processes: reaction of organic azides with group-XIII Lewis acids

Dichloroindium hydride revealed to be a valid alternative to tributyltin hydride for radical reduction of organic (alkyl, aryl, acyl, solfonyl) azides. The new approach entails mild reaction conditions and provides high yields of the corresponding amines and amides, also showing high degrees of selectivity. The system dichloroindium hydride / azides can be utilised in fivemembered ring closures of g-azidonitriles, as a new source of aminyl radicals for the attractive synthesis of interesting amidine compounds in the absence of both toxic reagents and tedious purification procedures. Allylindium dichloride seems a good substitute for dichloroindium hydride for generation of indium centred radicals under photolytic conditions, since it allows allylation of electrophilic azides (e.g. phenylsulfonyl azide) and halogen or ester δ-substituted azides, the latter through a 1,5-H transfer rearrangement mechanism. Evidences of the radical nature of the reactions mechanism were provided by ESR spectroscopy, furthermore the same technique, allowed to discover that the reaction of azides with indium trichloride and other group XIII Lewis acids, in particular gallium trichloride, gives rise to strongly coloured, persistent paramagnetic species, whose structure is consistent with the radical cation of the head-to-tail dimer of the aniline corresponding to the starting azide.

New Synthetic Polyamines as Multi-Target-Directed Ligands for the Treatment of Alzheimer's Disease and Cancer: Design, Synthesis and Biological Evaluation

Alzheimer's disease (AD) and cancer represent two of the main causes of death worldwide. They are complex multifactorial diseases and several biochemical targets have been recognized to play a fundamental role in their development. Basing on their complex nature, a promising therapeutical approach could be represented by the so-called "Multi-Target-Directed Ligand" approach. This new strategy is based on the assumption that a single molecule could hit several targets responsible for the onset and/or progression of the pathology. In particular in AD, most currently prescribed drugs aim to increase the level of acetylcholine in the brain by inhibiting the enzyme acetylcholinesterase (AChE). However, clinical experience shows that AChE inhibition is a palliative treatment, and the simple modulation of a single target does not address AD aetiology. Research into newer and more potent anti-AD agents is thus focused on compounds whose properties go beyond AChE inhibition (such as inhibition of the enzyme β-secretase and inhibition of the aggregation of beta-amyloid). Therefore, the MTDL strategy seems a more appropriate approach for addressing the complexity of AD and may provide new drugs for tackling its multifactorial nature. In this thesis, it is described the design of new MTDLs able to tackle the multifactorial nature of AD. Such new MTDLs designed are less flexible analogues of Caproctamine, one of the first MTDL owing biological properties useful for the AD treatment. These new compounds are able to inhibit the enzymes AChE, beta-secretase and to inhibit both AChE-induced and self-induced beta-amyloid aggregation. In particular, the most potent compound of the series is able to inhibit AChE in subnanomolar range, to inhibit β-secretase in micromolar concentration and to inhibit both AChE-induced and self-induced beta-amyloid aggregation in micromolar concentration. Cancer, as AD, is a very complex pathology and many different therapeutical approaches are currently use for the treatment of such pathology. However, due to its multifactorial nature the MTDL approach could be, in principle, apply also to this pathology. Aim of this thesis has been the development of new molecules owing different structural motifs able to simultaneously interact with some of the multitude of targets responsible for the pathology. The designed compounds displayed cytotoxic activity in different cancer cell lines. In particular, the most potent compounds of the series have been further evaluated and they were able to bind DNA resulting 100-fold more potent than the reference compound Mitonafide. Furthermore, these compounds were able to trigger apoptosis through caspases activation and to inhibit PIN1 (preliminary result). This last protein is a very promising target because it is overexpressed in many human cancers, it functions as critical catalyst for multiple oncogenic pathways and in several cancer cell lines depletion of PIN1 determines arrest of mitosis followed by apoptosis induction. In conclusion, this study may represent a promising starting pint for the development of new MTDLs hopefully useful for cancer and AD treatment.

Development of new therapeutic approaches in the treatments of Inflammatory Bowel Diseases: functional food and nutraceuticals vs synthetic peptides based vaccines

Inflammatory Bowel Diseases (IBD) are intestinal chronic relapsing diseases which ethiopathogenesis remains uncertain. Several group have attempted to study the role of factors involved such as genetic susceptibility, environmental factors such as smoke, diet, sex, immunological factors as well as the microbioma. None of the treatments available satisfy several criteria at the same time such as safety, long-term remission, histopatological healing, and specificity. We used two different approaches for the development of new therapeutic treatment for Inflammatory Bowel Disease. The first is focused on the understanding of the potential role of functional food and nutraceuticals nutrients in the treatment of IBD. To do so, we investigated the role of Curcuma longa in the treatment of chemical induced colitis in mice model. Since Curcma Longa has been investigated for its antinflammatory role related to the TNFα pathway as well investigators have reported few cases of patients with ulcerative colites treated with this herbs, we harbored the hypothesis of a role of Curcuma Longa in the treatment f IBD as well as we decided to assess its role in intestinal motility. The second part is based on an immunological approach to develop new drugs to induce suppression in Crohn’s disease or to induce mucosa immunity such as in colonrectal tumor. The main idea behind this approach is that we could manipulate relevant cell-cell interactions using synthetic peptides. We demonstrated the role of the unique interaction between molecules expressed on intestinal epithelial cells such as CD1d and CEACAM5 and on CD8+ T cells. In normal condition this interaction has a role for the expansion of the suppressor CD8+ T cells. Here, we characterized this interaction, we defined which are the epitope involved in the binding and we attempted to develop synthetic peptides from the N domain of CEACAM5 in order to manipulate it.

New synthetic bile acid analogue agonists of FXR and TGR5 receptors: Analytical methodologies for the study of their physico-chemical properties, pharmacokinetic activity and metabolism.

This thesis reports an integrated analytical approach for the study of physicochemical and biological properties of new synthetic bile acid (BA) analogues agonists of FXR and TGR5 receptors. Structure-activity data were compared with those previous obtained using the same experimental protocols on synthetic and natural occurring BA. The new synthetic BA analogues are classified in different groups according also to their potency as a FXR and TGR5 agonists: unconjugated and steroid modified BA and side chain modified BA including taurine or glycine conjugates and pseudo-conjugates (sulphonate and sulphate analogues). In order to investigate the relationship between structure and activity the synthetic analogues where admitted to a physicochemical characterization and to a preliminary screening for their pharmacokinetic and metabolism using a bile fistula rat model. Sensitive and accurate analytical methods have been developed for the quali-quantitative analysis of BA in biological fluids and sample used for physicochemical studies. Combined High Performance Liquid Chromatography Electrospray tandem mass spectrometry with efficient chromatographic separation of all studied BA and their metabolites have been optimized and validated. Analytical strategies for the identification of the BA and their minor metabolites have been developed. Taurine and glycine conjugates were identified in MS/MS by monitoring the specific ion transitions in multiple reaction monitoring (MRM) mode while all other metabolites (sulphate, glucuronic acid, dehydroxylated, decarboxylated or oxo) were monitored in a selected-ion reaction (SIR) mode with a negative ESI interface by the following ions. Accurate and precise data where achieved regarding the main physicochemical properties including solubility, detergency, lipophilicity and albumin binding . These studies have shown that minor structural modification greatly affect the pharmacokinetics and metabolism of the new analogues in respect to the natural BA and on turn their site of action, particularly where their receptor are located in the enterohepatic circulation.

Bioconjugation and synthetic approach towards enantioenriched gem-difluoromethylene compounds through carbenium ions

Bioconjugation of peptides and asymmetric synthesis of gem-difluoromethylene compounds are areas of the modern organic chemistry for which mild and selective methods continue to be developed. This thesis reports new methodologies for these two areas based on the use of stabilized carbenium ions. The reaction that makes the bioconjugation of peptides possible takes place via the direct nucleophilic substitution of alcohols and is driven by the spontaneous formation of stabilized carbenium ions in water. By reacting with the thiol group of cysteine in very mild conditions and with a high selectivity, these carbenium ions allow the site-specific ligation of polypeptides containing cysteine and their covalent derivatization with functionalized probes. The ligation of the indole ring of tryptophan, an emerging target in bioconjugation, is also shown and takes place in the same conditions. The second area investigated is the challenging access to optically active gem-difluoromethylene compounds. We describe a methodology relying on the synthesis of enantioenriched 1,3-benzodithioles intermediates that are shown to be precursors of the corresponding gem-difluoromethylene analogues by oxidative desulfurization-fluorination. This synthesis takes advantage of the highly enantioselective organocatalytic α-alkylation of aldehydes with the benzodithiolylium ion and of the wide possibilities of synthetic transformations offered by the 1,3-benzodithiole group. This approach allows the asymmetric access to complex gem-difluoromethylene compounds through a late-stage fluorination step, thus avoiding the use of fluorinated building blocks.

Fast and accurate numerical solutions in some problems of particle and radiation transport: synthetic acceleration for the method of short characteristics, Doppler-broadened scattering kernel, remote sensing of the cryosphere

The aim of this work is to present various aspects of numerical simulation of particle and radiation transport for industrial and environmental protection applications, to enable the analysis of complex physical processes in a fast, reliable, and efficient way. In the first part we deal with speed-up of numerical simulation of neutron transport for nuclear reactor core analysis. The convergence properties of the source iteration scheme of the Method of Characteristics applied to be heterogeneous structured geometries has been enhanced by means of Boundary Projection Acceleration, enabling the study of 2D and 3D geometries with transport theory without spatial homogenization. The computational performances have been verified with the C5G7 2D and 3D benchmarks, showing a sensible reduction of iterations and CPU time. The second part is devoted to the study of temperature-dependent elastic scattering of neutrons for heavy isotopes near to the thermal zone. A numerical computation of the Doppler convolution of the elastic scattering kernel based on the gas model is presented, for a general energy dependent cross section and scattering law in the center of mass system. The range of integration has been optimized employing a numerical cutoff, allowing a faster numerical evaluation of the convolution integral. Legendre moments of the transfer kernel are subsequently obtained by direct quadrature and a numerical analysis of the convergence is presented. In the third part we focus our attention to remote sensing applications of radiative transfer employed to investigate the Earth's cryosphere. The photon transport equation is applied to simulate reflectivity of glaciers varying the age of the layer of snow or ice, its thickness, the presence or not other underlying layers, the degree of dust included in the snow, creating a framework able to decipher spectral signals collected by orbiting detectors.

Semi-synthetic bile acids as novel drug candidate in liver diseases: physico-chemical characterization and HPLC-ES-MS/MS methods for their quali-quantitative analysis in different experimental animal models

The physico-chemical characterization, structure-pharmacokinetic and metabolism studies of new semi synthetic analogues of natural bile acids (BAs) drug candidates have been performed. Recent studies discovered a role of BAs as agonists of FXR and TGR5 receptor, thus opening new therapeutic target for the treatment of liver diseases or metabolic disorders. Up to twenty new semisynthetic analogues have been synthesized and studied in order to find promising novel drugs candidates. In order to define the BAs structure-activity relationship, their main physico-chemical properties (solubility, detergency, lipophilicity and affinity with serum albumin) have been measured with validated analytical methodologies. Their metabolism and biodistribution has been studied in “bile fistula rat”, model where each BA is acutely administered through duodenal and femoral infusion and bile collected at different time interval allowing to define the relationship between structure and intestinal absorption and hepatic uptake ,metabolism and systemic spill-over. One of the studied analogues, 6α-ethyl-3α7α-dihydroxy-5β-cholanic acid, analogue of CDCA (INT 747, Obeticholic Acid (OCA)), recently under approval for the treatment of cholestatic liver diseases, requires additional studies to ensure its safety and lack of toxicity when administered to patients with a strong liver impairment. For this purpose, CCl4 inhalation to rat causing hepatic decompensation (cirrhosis) animal model has been developed and used to define the difference of OCA biodistribution in respect to control animals trying to define whether peripheral tissues might be also exposed as a result of toxic plasma levels of OCA, evaluating also the endogenous BAs biodistribution. An accurate and sensitive HPLC-ES-MS/MS method is developed to identify and quantify all BAs in biological matrices (bile, plasma, urine, liver, kidney, intestinal content and tissue) for which a sample pretreatment have been optimized.