Synthesis and applications of chiral catalysts based on heterocyclic units

The present PhD Thesis was mainly focused on the synthesis and the applications of chiral catalysts based on heterocyclic units.
As an appendix of the manuscript, the work concerning my abroad period spent at the University of Glasgow is reported, whose research topic was the synthesis of benzoxazoles through a one-pot two-step procedure involving the use of first iron(III) and copper(I) catalysis for the second step. The fields of catalysis investigated are the Lewis Base, the Brønsted acid catalysis and finally the catalysis promoted by phosphoramidites. As a contribution for the stereoselective Lewis Base catalysis, the synthesis and the resolution of TetraPh-Tol-BITIOPO (Chapter 1, 37) was carried out, whose application can be considered a further investigation of the huge potentialities of the 3,3’-bithiophenic enantiopure TetraMe-BITIOPO in Lewis-base catalysed Lewis-acid mediated reactions. For what concern Brønsted acid catalysis, a new chiral phosphoric acid based on a decahydroquinoxalinic scaffold bearing in the position 2 and 3 two thienylic units (Chapter 2, 19) was synthesized and tested in two preliminary stereoselective organocatalytic transformations. Based on the same decahydroquinoxalinic backbone, we decided to design a new heterocyclic- based diphosphinoxide (Chapter 2, 42). In Chapter 3, a new heterocyclic phosphoramidite based on a 3,3’-bithiophenic scaffold (L6) was synthesized and employed in combination with a copper salt in some catalytic stereoselective 1,4- additions of diethyl zinc to enones and trisubstituted nitroalkenes

Hyaluronan: a new player in the modulation of adaptive changes to neuromuscular damage in the gastrointestinal tract

Pathological conditions of the gastrointestinal tract, such as chronic inflammatory states or intestinal ischemia/reperfusion (I/R) injury, have severe consequences on different cell types constituting the enteric microenvironment. In particular, myenteric neurons are especially sensitive, and can be irreversibly damaged, resulting in structural and functional changes of the enteric circuitries. Such changes may be, at least in part, due to the interplay among different cell populations of enteric microenvironment. Hyaluronan (HA) represents an important molecule of the extracellular matrix (ECM), which provides an significant framework for that microenvironment. In this context, the aim of my thesis was to evaluate possible changes of HA homeostasis in myenteric ganglia after an experimentally induced colitis and an in vivo-induced ischemia/reperfusion (I/R) damage in rats. Results showed that myenteric neurons synthesize HA to form a well-structured perineuronal net, which undergoes derangement when myenteric ganglia homeostasis is perturbed, i.e. during the inflammatory state in the experimentally-induced colitis. In addition, data indicated that in the neuromuscular compartment of the rat small intestine, an I/R injury increases HA levels, and that HA may influence both the excitatory and inhibitory components of the peristaltic reflex. Overall, this study provides evidence that HA deposition within myenteric ganglia may have a homeostatic role, contributing to the control of myenteric neuron structure and function and supporting the efficiency of the gastrointestinal transit. Hence, modulation of HA deposition within myenteric ganglia may ameliorate intestinal motility patterns related to these disease states

The Peptide Transporters of teleost fish, an emerging model in translational research: functional characterization and comparative study of SLC15A1a (PepT1a) and SLC15A1b (PepT1b) transporters

Translational research is the process that applies knowledge from basic biology to techniques and devices to solve critical medical issues. The aim of basic and translational research is to identify specific cellular and animal models for every single process, physiological or pathological, under study. To find out the adequate animal model, one of the best approaches is to study the orthologues of human genes in different species. The increase in the number of fully sequenced genomes offers important advances in comparative genomics and in translational research. The finding that in zebrafish genome there are orthologues for most human genes is particularly important. In fact, zebrafish are for many aspects an outstanding model organism for high throughput phenotyping and modeling human disease and disorders. The aim of the research work of this thesis is to understand and compare the function of proteins belonging to the SoLute Carrier family 15 member A1 (PepT1) in different fish orthologues. The intestinal transporter PepT1 represents a major route of peptides and drug intake. Besides the important role of PepT1 in the nutrient uptake and sensing, the study of the functional properties of this transporter is of great importance not only for the involvement in pathological states, but also for its double role in therapeutic approaches. Recently, PepT1 has become an attractive target for potential therapeutic models for developing and enhancing drug-delivery systems

Genetic abnormalities as diagnostic and prognostic markers in B cell lymphomas: role of new molecular technologies in personalized medicine for extranodal diffuse large B cell lymphoma (EN-DLBCL) and follicular lymphoma (FL)

A plethora of molecular biomarkers are available nowadays in the field of cancer research. However, it is crucial to understand when and how they can be integrated into the clinical setting, translating experimental results from bench to bedside, with the aim of improving patients’ care. We decided to investigate the role of some of these biomarkers in two subtypes of non-Hodgkin lymphoma which still represent a challenge for both researchers and clinicians. We started from diffuse large B cell lymphomas (DLBCLs), investigating a multicentric series of primary extranodal DLBCLs. Overall, data analysis provided strong evidence that the distribution of immunophenotypic, cytogenetic and survival characteristics is site-dependent. We next moved to follicular lymphoma (FL). The translocation (14;18), leading to BCL2 protein overexpression, is considered the genetic hallmark of FL. We tested the incidence of BCL2 negative FLs in a series of Italian patients from the Insubric region, concluding that BCL2 rearrangement in FL is not as frequent as generally reported and that the genetic landscape of FL is more complex than previously thought. What we learned is that even within an individual clinical entity, there is considerable heterogeneity with respect to genetic alterations, expression of commonly assayed markers and, most important, outcome. The personalized approach acknowledges this complexity and gives us tools for the continuous improvement of patients’ care

Statistical analysis of Dynamic Light Scattering data: from the Schätzel formulas to new approaches based on multi−tau Photon Counting Histogram and variance methods

Dynamic Light Scattering (DLS) is an optical technique aimed at the determination of the dimensions of small particles in a suspension in a range of diameters from about 1 nm to 1 μm. It consists in the measurement, at a fixed angle, of the scattered intensity radiation that, because of the Brownian motion of the particles, fluctuates stochastically with time. A DLS experiment provides the correlation function of the received intensity which is dependent from the coherence time of the fluctuations, that is related to the hydrodynamical radius of the particles that constitute the sample. In this thesis work, we analyzed the error bars associated to a correlation function and proposed a new approach for the analysis of Dynamic Light Scattering data based on multi-tau Photon Counting Histogram and variance methods. We obtained the following results and future perspectives. Statistical analysis of Dynamic Light Scattering data The first goal of this work consists in the analytical determination of the error bars associated to a correlation function. This problem has been analyzed by K. Schätzel in the 1990s. He provided two analytical expressions for the covariance matrix and the variance for the specific case of a Lorentzian spectrum where the correlation function is characterized by a single exponential decay. These formulas do not include the effects due to a triangular averaging and are consequently inaccurate for sampling times higher or similar than the coherence time. In this work, we analyzed these formulas and worked out two exact analytical expressions in which the effects due to the triangular averaging are corrected to all orders. By the use of extensive computer simulations and experimental test carried out on dilute dispersions of calibrated latex spheres, we have shown that the new formula for the variance works quite accurately for sampling times both higher and lower than the coherence time and can be applied well beyond the specific case of a single exponential decay auto-correlation function. We believe that these new covariance and variance formulas would turn out to be a fairly useful tool for the wide community of scientists working in the field of DLS. Nowadays, this technique is ubiquitously based on the use of multi-tau correlators, where, if not properly taken into account, the triangular averaging would introduce huge errors in the estimates of the uncertainties to be associated to the measured correlation function. Photon Counting Histogram applied for the analysis of Dy- namic Light Scattering data The second goal of this thesis work consists in the analysis of a second method for performing particle sizing. In this case the coherence time of the intensity fluctuations will not be recovered from a correlation, but from a series of histograms of the photon counts detected over different integration times. It represents, once normalized, the probability distribution to detect a discrete number of photon counts over a given sam- pling time. In my work we applied the Photon Counting Histogram (PCH) technique for the analysis of DLS data and we analyzed, for a monodisperse sample, the combined use of these two techniques and the advantages it gives with respect to a single one. This technique has been developed for the first time by E. Gratton for the analysis of fluorescent data. His theory works when the sampling time is much smaller than the coherence time. By using the PCH method for the analysis of DLS data, because of the raw data has been taken with the multi-tau method, we analyzed different histograms generated with different sampling times which are both higher and lower than the co- herence time. Moreover, we exploited an alternative method to perform particle sizing by recovering the hydrodynamical diameter by fitting not the histograms themselves, but the variances of the photon count distributions recovered stage by stage as a func- tion of the sampling time. We believe that both the PCH method and the variance analysis applied to DLS data may be useful because they provide a different technique to perform particle sizing, which can work independently from the auto- (or cross-) correlation functions. Both these three techniques can work in parallel and, if they provide the same result, it can be a further proof or cross-check for the correctness of the recovered hydrodynamical diameter of the considered sample

Adolescent Δ9-tetrahydrocannabinol exposure differently affects histone modifications in the brain of female and male rats

Despite the increasing evidence of a possible interaction between adolescent Cannabis abuse and the subsequent development of psychiatric disorders, Cannabis remains the illicit drug most abused by adolescents. We have previously demonstrated that female rats chronically treated during adolescence with increasing doses of delta-9-tetrahydrocannabinol (THC), the main psychoactive ingredient of cannabis, develop a depressive/psychotic-like phenotype in adulthood. Interestingly, only chronic adolescent exposure to THC, but not adult exposure, led to this complex phenotype, suggesting that adolescence may represent a more vulnerable period for the adverse effect of THC. However, the neurobiology of this vulnerability is not still clear. Considering the important role assumed by epigenetics in the etiopathogenesis of psychiatric disorders, the main goal of this thesis is to extend our knowledge on the impact of adolescent THC exposure on histone modifications occurring in other brain areas involved in the different aspects of the depressive/psychotic-like phenotype described in our animals. Specifically, we considered the Hippocampus for its involvement in cognition, the Nucleus Accumbens for its role in the reward circuit, and the Amygdala for its relevance in the emotional behaviour. To investigate the existence of age-specificity of THC effects, we performed the same analysis also after adult THC treatment. To investigate sex-dependency of THC response, we also checked THC response in adolescent male animals. First of all, adolescent (PND 35-45) and adult (PND 75-85) female Sprague-Dawley rats were treated twice a day with increasing intraperitoneal (ip) doses of THC: 2.5 mg/kg, 5 mg/kg, and 10 mg/kg or with its vehicle. Two, 24 and 48 hours after the end of the treatment, the brain areas of interest were collected and. Histone modifications associated with both transcriptional repression (H3K9 di- and tri-methylation, H3K27 trimethylation) and activation (H3K9 and H3K14 acetylation) were evaluated. Chronic THC exposure affected histone modifications in the brain of female rats in a region- and age-specific manner. Indeed, THC acted on different targets depending on the considered brain areas and, remarkably, the adolescent brain was generally more sensitive to THC exposure compared to the adult one. Specifically, in the Hippocampus of adolescent rats, THC induced a reduction of H3K14ac levels 2 hours after the end of the treatment. This was followed by a significant increase in di- and tri-methylation of H3K9 at 24 hours. Regarding the Nucleus Accumbens, H3K9me3 was significantly increased 2 hours after the end of the treatment. This enhancement was maintained 24 hours later, and it was paralleled by a significant increase in H3K9me2 and H3K14ac levels. On the contrary, at 48h, H3K9me3 levels, as well as H3K9me2 and H3K14ac levels were significantly reduced. In the Amygdala, THC administration induced a significant increase in H3K9me2 levels 2 hours after the end of the treatment. Twenty-four hours later, while this alteration returned to control values, H3K9me3 levels were significantly enhanced. Adult female rats exposed to chronic THC showed a different pattern of histone alterations. In the Hippocampus and Nucleus Accumbens, H3K14 acetylation levels were significantly increased, respectively, 2 and 24 hours after the end of the treatment. Intriguingly, a more complex picture is present in the adult Amygdala, in which a significant decrease in H3K9me2 and H3K27me3 were induced immediately after the cease of the treatment. Twenty-four hours later H3K9ac was significantly reduced, and at 48 hours, H3K14ac levels were significantly decreased. As a whole, the investigation performed in female rats suggests that in the adolescent brain THC induced a primary effect represented by changes leading to transcriptional repression, whereas the primary effect induced by adult THC exposure led to transcriptional activation. Interestingly, only in the adolescent brain, the primary effect was followed by a homeostatic response to counterbalance the THC-induced repressive effect, except in the amygdala. The presence of a more complex response in the adolescent brain may be part of the mechanisms that make the adolescent brain vulnerable to THC adverse effects. The second aim of this thesis was to extend our knowledge on the impact of adolescent THC exposure on histone modifications occurring in different brain areas of male rats. To this aim, adolescent (PND 35-45) male Sprague-Dawley rats were treated with the same protocol previously described for females and we conducted the same analysis in the Prefrontal Cortex, Hippocampus and Nucleus Accumbens. Chronic THC exposure affected histone modifications in the brain of male rats in a region- specific manner. Surprisingly, in the Prefrontal cortex and Hippocampus, we did not found any histone alterations at any intervals of time, and only in the Nucleus Accumbens we found significant alterations in H3K9me3 levels. Specifically, H3K9me3 was decreased immediately after the end of the treatment and then increased 24h later. Further studies are needed to clarify the epigenetic landscape in the brain of male rats and how it could account for the development of the psychotic-like phenotype described in these animals. However, it is possible to conclude that Cannabis abuse during adolescence could impair the brain network functionality acting through a mechanism involving histone modifications that is characterized by sex-specificity

Neutrino emission from Blazars.

During the last decade, multi-messenger astronomy has become increasingly relevant for the astrophysical community. In this context the discovery of PeV neutrinos by IceCube in 2012, in clear excess to the expected atmospheric flux at very-high energy (≳ 100 TeV ), marked the beginning of the high-energy neutrino astrophysics era. Differently from photons, neutrinos can carry information about the core of the astrophysical objects that produce them, giving us a better understanding of the internal composition of their sources. The origin of these neutrinos is still an open issue. Among the possible extragalactic neutrino sources, Blazars start to stand out. Blazars are associated to active galactic nuclei hosting a relativistic jet oriented close to our line of sight. The presence of jets and the strong non-thermal emission up to the TeV band makes them natural accelerators of particles. In September 2017 the spatial coincidence between a neutrino event detected by IceCube, with a good angular resolution, and a blazar, TXS 0506+056, was observed for the rst time. This event is even more intriguing because of the high-state emission in γ-ray band of this blazar. During the three years of my PhD, I focused my attention on the study of blazar objects as neutrino emitters. In this thesis all my work on this topic is presented

Research on a bright light source: optics, technology and effects on humans

There are countless reasons why the Sun is essential: one of these is daylight. This light, that comprises both the component diffused by the atmosphere and the transmitted one, is the reference for a naturally lit scene, also being the main synchronizer of our circadian rhythms. I carried out my research in the framework of two European projects (COELUX and SKYCOAT), coordinated by the University of Insubria, which were related to the reconstruction of natural light indoors. In such a context, in collaboration with the consortium partners but mainly with the spin off CoeLux Srl, my research dealt with the bright light source used to artificially recreate the sun, regarding the optical design, the technological implementation and the effects produced on humans. The approach is interdisciplinary, involving different branches of science as well as technology, psychology and business perspectives. These aspects were analyzed, insofar as relevant, in order to design, optimize, and realize the prototypes. The actions that were carried out and are reported in this work comprise: the definition of the requirements for creating a valid light source, a review of the state of the art related to the areas of interest, the development of a solution meeting all the requirements and the characterization and testing of the prototypes. The LED-based light projector that includes the results of this work perfectly suits the CoeLux technology and is a cornerstone in the study of effects on humans

Development of saporin-based therapeutic options for the treatment of solid tumors.

Up to now, cancer represents one of the most challenging disease to treat, mostly due to its ability to adapt and negatively respond to current available therapies. Over the past 30 years, Ribosome Inactivating Proteins (RIPs) have attracted great interest in the scientific community for their therapeutic potential. Indeed, such toxins have been extensively used as potent and versatile therapeutic weapons due to important advantages compared to chemotherapeutics: for instance, they act independently form cell cycle, killing both quiescent and dividing cells, limiting the development of cancer resistance. However, the successful application of toxins-based therapeutics to solid tumors remains to be demonstrated. In this study, we explored the potential use of the plant-RIP saporin (SAP) for the treatment of solid tumors. In particular, we propose two different strategies, in which SAP is selectively and safely conveyed to malignant cells either in the form of recombinant protein through genetically fused targeting moieties, or throughout cell-derived extracellular vesicles as vehicles. Overall our data indicate that SAP-based recombinant proteins are promising antitumoral therapeutic options. Applied as single or combined treatment, as well as used together with traditional therapeutics, they appropriately address both intra and inter- tumor heterogeneity. In addition, the use of exosomes as SAP nanocarriers is a promising strategy to improve safety and drug delivery to tumor cells

La “buona morte”: analisi del profilo storico e ruolo delle cure palliative nell’accompagnamento di fine vita

This research focuses on the link between the concept of “good death” and the notion of euthanasia, words connected to each other from an etymological point of view but which differ in meaning and historical evolution. The analysis of the sources relating to “good death” has shown three main research macro-groups: one relating to the concept of “good death” in the ancient world from the classical antiquity to the late imperial age; one relative to the link between “good death” and palliative care and, lastly, the role that consolation plays in the accompaniment of the terminally ill. These reference passages have been chosen on the basis of three useful tools in the setting of Greek-Latin Philology and of ancient Christian Literature: the Thesaurus Linguae Graecae, the Thesaurus Linguae Latinae and the Bibleworks. The analysis of these passages has revealed that the notion of euthanasia in classical Greek and Latin conveyed a concept very different from the modern one. Two fundamental concepts were observed: that of “good death” and that of suicide. In Antiquity the three concepts of euthanasia, “good death” and suicide do not appear any different to today, on the contrary, the notions of euthanasia and “good death” permeate each other and euthanasia, as well as suicide often appear in the sources analyzed as “good death”. The point of view held by the professionals operating in the sector, which has been investigated in the second chapter in the form of an interview, reveals the importance of palliative care in accompanying the suffering person in the final stage of life. The third and final chapter, investigates the role of consolation both in Christian literature (The Bible; Greek and Latin Church Fathers) as well as in the concrete reality of care giving today through the analysis of the results of a survey carried out on caregivers of ten Italian hospices