Polymorphs solvates and co-crystals of molecular materials

The scope of my research project is to produce and characterize new crystalline forms of organic compounds, focusing the attention on co-crystals and then transferring these notions on APIs to produce co-crystals of potential interest in the pharmaceutical field. In the first part of this work co-crystallization experiments were performed using as building blocks the family of aliphatic dicarboxylic acids HOOC-(CH2)n-COOH, with n= 2-8. This class of compounds has always been an object of study because it is characterized by an interesting phenomenon of alternation of melting points: the acids with an even number of carbon atoms show a melting point higher than those with an odd one. The acids were co-crystallized with four dipyridyl molecules (formed by two pyridine rings with a different number of bridging carbon atoms) through the formation of intermolecular interactions N•••(H)O. The bases used were: 4,4’-bipyridine (BPY), 1,2-bis(4-pyridyl)ethane (BPA), 1,2-(di-4-pyridyl)ethylene (BPE) and 1,2-bis(4-pyridyl)propane (BPP). The co-crystals obtained by solution synthesis were characterized by different solid-state techniques to determine the structure and to see how the melting points in co-crystals change. In the second part of this study we tried to obtain new crystal forms of compounds of pharmaceutical interest. The APIs studied are: O-desmethylvenlafaxine, Lidocaine, Nalidixic Acid and Sulfadiazine. Each API was subjected to Polymorph Screening and Salt/Co-crystal Screening experiments to identify new crystal forms characterized by different properties. In a typical Salt/Co-crystal Screening the sample was made to react with a co-former (solid or liquid) through different methods: crystallization by solution, grinding, kneading and solid-gas reactions. The new crystal forms obtained were characterized by different solid state techniques (X-ray single crystal diffraction, X-ray powder diffraction, Differential Scanning Calorimetry, Thermogravimetric Analysis, Evolved gas analysis, FT-IR – ATR, Solid State N.M.R).

General effective medium model for the complex permittivity extraction with an open-ended coaxial probe in presence of a multilayer material under test

The work presented in this thesis is focused on the open-ended coaxial-probe frequency-domain reflectometry technique for complex permittivity measurement at microwave frequencies of dispersive dielectric multilayer materials. An effective dielectric model is introduced and validated to extend the applicability of this technique to multilayer materials in on-line system context. In addition, the thesis presents: 1) a numerical study regarding the imperfectness of the contact at the probe-material interface, 2) a review of the available models and techniques, 3) a new classification of the extraction schemes with guidelines on how they can be used to improve the overall performance of the probe according to the problem requirements.

Study of Venturia inaequalis sensitivity to fungicides through molecular and biological methodologies

This study was aimed to correlate the results of relative germination from in vitro tests by trifloxystrobin with those of qPCR on a wide range of V. inaequalis populations and monoconidial isolates. Samples were collected in Italian and Turkish distinct locations from orchards with different scab management. In this study, an allele-specific qPCR with primer sets designed was successfully developed to quantitatively determine the frequency of QoI-resistant allele G143A in populations and monoconidial isolates of V. inaequalis. qPCR followed a similar pattern to that obtained using in vitro conidial germination test in very sensitive and very resistant populations. However, the variability between two test results was observed in hetereogenous populations. Therefore, the results of correlations between in vitro and qPCR showed a positive but not very high correlation for Venturia inaequalis populations (R2=0.70). On the contrary, this correlation between two assessment methods was very high for monoconidial isolates (R2=0.92). Results obtained in quantitative PCR and from traditional spore germination assay differed for the same fungal population and in some cases, it is difficult to assess the resistance in the field by only qPCR. It was concluded that it is not always possible to correlate the frequency of detection of the mutation with biological assessment. In such situations, monitoring by molecular techniques must be supported by standard in vitro resistance assessments and observation of field performance in order to have correct conclusions.

Multimethodological study of molecular recognition phenomena

The study of the bio-recognition phenomena behind a biological process is nowadays considered a useful tool to deeply understand physiological mechanisms allowing the discovery of novel biological target and the development of new lead candidates. Moreover, understanding this kind of phenomena can be helpful in characterizing absorption, distribution, metabolism, elimination and toxicity properties of a new drug (ADMET parameters). Recent estimations show that about half of all drugs in development fail to make it to the market because of ADMET deficiencies; thus a rapid determination of ADMET parameters in early stages of drug discovery would save money and time, allowing to choose the better compound and to eliminate any losers. The monitoring of drug binding to plasma proteins is becoming essential in the field of drug discovery to characterize the drug distribution in human body. Human serum albumin (HSA) is the most abundant protein in plasma playing a fundamental role in the transport of drugs, metabolites and endogenous factors; so the study of the binding mechanism to HSA has become crucial to the early characterization of the pharmacokinetic profile of new potential leads. Furthermore, most of the distribution experiments carried out in vivo are performed on animals. Hence it is interesting to determine the binding of new compounds to albumins from different species to evaluate the reliability of extrapolating the distribution data obtained in animals to humans. It is clear how the characterization of interactions between proteins and drugs determines a growing need of methodologies to study any specific molecular event. A wide variety of biochemical techniques have been applied to this purpose. High-performance liquid affinity chromatography, circular dichroism and optical biosensor represent three techniques that can be able to elucidate the interaction of a new drug with its target and with others proteins that could interfere with ADMET parameters.

Innovative techniques for sport turf managing: Agronomic and phyisiological implications

The growing substrate of the putting greens is considered a key factor for a healthy turf ecosystem. Actually detailed study on the effects of growth promoting bacteria and biostimulants on a professional sport turf are very limited. This thesis aimed to study the effectiveness of different microorganisms and biostimulants in order to improve the knowledge relative to the relationship between the beneficial microflora and root apparatus of sport turfs. The research project was divided in three principal steps: Initially, commercial products based on biostimulants and microorganisms were tested on a Lolium perenne L. essence grown in a controlled-environment. The principal evaluations were the study of the habitus of plants, biomass production and length of leaves and roots. Were studied the capacity of colonization of microorganisms within root tissues and rhizosphere. In the second step were developed two different biostimulant solutions based on effective microorganisms, mycorrhizae and humic acids. This test was conducted both on an Agrostis stolonifera putting green (Modena Golf & Country Club) in a semi-field condition and within a growth chamber on a Lolium perenne L. essence. Fungicide and chemicals applications were suspended in order to assess the effectiveness of the inoculants for nutrition and control of pests. In the last step, different microorganism mixes and biostimulants were tested on an experimental putting green in the Turf Research Center (TRC) (Virginia Tech, United States) in a real managing situation. The effects of different treatments were studied maintaining all chemicals and mechanicals managements scheduled during a sport season. Both growth-chamber and field results confirmed the capacity of microorganisms based biostimulants to promote the physiologic conditions of the plants, improve the growth of the roots and enhance the aesthetic performance of the turf. Molecular analysis confirmed the capacity of microorganisms to colonize the root tissues.

In-situ and real time scanning probe microscopy of organic ultra thin films

In recent decades, Organic Thin Film Transistors (OTFTs) have attracted lots of interest due to their low cost, large area and flexible properties which have brought them to be considered the building blocks of the future organic electronics. Experimentally, devices based on the same organic material deposited in different ways, i.e. by varying the deposition rate of the molecules, show different electrical performance. As predicted theoretically, this is due to the speed and rate by which charge carriers can be transported by hopping in organic thin films, transport that depends on the molecular arrangement of the molecules. This strongly suggests a correlation between the morphology of the organic semiconductor and the performance of the OTFT and hence motivated us to carry out an in-situ real time SPM study of organic semiconductor growth as an almost unprecedent experiment with the aim to fully describe the morphological evolution of the ultra-thin film and find the relevant morphological parameters affecting the OTFT electrical response. For the case of 6T on silicon oxide, we have shown that the growth mechanism is 2D+3D, with a roughening transition at the third layer and a rapid roughening. Relevant morphological parameters have been extracted by the AFM images. We also developed an original mathematical model to estimate theoretically and more accurately than before, the capacitance of an EFM tip in front of a metallic substrate. Finally, we obtained Ultra High Vacuum (UHV) AFM images of 6T at lying molecules layer both on silicon oxide and on top of 6T islands. Moreover, we performed ex-situ AFM imaging on a bilayer film composed of pentacene (a p-type semiconductor) and C60 (an n-type semiconductor).

Development of new molecular methods for the diagnosis and the study of viral diseases of fish

The increase in aquaculture operations worldwide has provided new opportunities for the transmission of aquatic viruses. The occurrence of viral diseases remains a significant limiting factor in aquaculture production and for the sustainability. The ability to identify quickly the presence/absence of a pathogenic organism in fish would have significant advantages for the aquaculture systems. Several molecular methods have found successful application in fish pathology both for confirmatory diagnosis of overt diseases and for detection of asymptomatic infections. However, a lot of different variants occur among fish host species and virus strains and consequently specific methods need to be developed and optimized for each pathogen and often also for each host species. The first chapter of this PhD thesis presents a complete description of the major viruses that infect fish and provides a relevant information regarding the most common methods and emerging technologies for the molecular diagnosis of viral diseases of fish. The development and application of a real time PCR assay for the detection and quantification of lymphocystivirus was described in the second chapter. It showed to be highly sensitive, specific, reproducible and versatile for the detection and quantitation of lymphocystivirus. The use of this technique can find multiple application such as asymptomatic carrier detection or pathogenesis studies of different LCDV strains. The third chapter, a multiplex RT-PCR (mRT-PCR) assay was developed for the simultaneous detection of viral haemorrhagic septicaemia (VHS), infectious haematopoietic necrosis (IHN), infectious pancreatic necrosis (IPN) and sleeping disease (SD) in a single assay. This method was able to efficiently detect the viral RNA in tissue samples, showing the presence of single infections and co-infections in rainbow trout samples. The mRT-PCR method was revealed to be an accurate and fast method to support traditional diagnostic techniques in the diagnosis of major viral diseases of rainbow trout.

Computational Investigation of the Molecular Mechanisms Regulating Nucleic Acid Processing Metalloenzymes

Polymerases and nucleases are enzymes processing DNA and RNA. They are involved in crucial processes for cell life by performing the extension and the cleavage of nucleic acid chains during genome replication and maintenance. Additionally, both enzymes are often associated to several diseases, including cancer. In order to catalyze the reaction, most of them operate via the two-metal-ion mechanism. For this, despite showing relevant differences in structure, function and catalytic properties, they share common catalytic elements, which comprise the two catalytic ions and their first-shell acidic residues. Notably, recent studies of different metalloenzymes revealed the recurrent presence of additional elements surrounding the active site, thus suggesting an extended two-metal-ion-centered architecture. However, whether these elements have a catalytic function and what is their role is still unclear. In this work, using state-of-the-art computational techniques, second- and third-shell elements are showed to act in metallonucleases favoring the substrate positioning and leaving group release. In particular, in hExo1 a transient third metal ion is recruited and positioned near the two-metal-ion site by a structurally conserved acidic residue to assist the leaving group departure. Similarly, in hFEN1 second- and third-shell Arg/Lys residues operate the phosphate steering mechanism through (i) substrate recruitment, (ii) precise cleavage localization, and (iii) leaving group release. Importantly, structural comparisons of hExo1, hFEN1 and other metallonucleases suggest that similar catalytic mechanisms may be shared by other enzymes. Overall, the results obtained provide an extended vision on parallel strategies adopted by metalloenzymes, which employ divalent metal ion or positively charged residues to ensure efficient and specific catalysis. Furthermore, these outcomes may have implications for de novo enzyme engineering and/or drug design to modulate nucleic acid processing.

Towards the conservation of vulnerable marine large predators: morphometric, molecular and microchemical variation of historical sawfish rostra from Mediterranean collections

Sawfishes (Chondrichthyes, Pristidae) are considered one of the most endangered families among elasmobranchs. Extensive efforts are required worldwide to gather solid information on historical and recent changes in the composition/range of species. In this study, we have implemented an integrative approach to characterize the species diversity and the abundance of historical rostra of sawfishes from museums and private collections of the Mediterranean area. The identification at the species level of 172 dried rostra was carried out through the integration of both traditional and geometric morphometric techniques with molecular tools, allowing the assessment of a robust methodical approach to discriminate species. In addition, we analysed 35 rostral teeth to clarify the past distribution of sawfish species considering the isotopic composition of oxygen and carbon. The morphometric, molecular, and geographical characterization of samples was accompanied by the preliminary evaluation of growth structures and the inspection of the strontium isotope composition in two teeth to unravel movement patterns of individuals across different salinities of water. Results were integrated with currently available data from public repositories and showed that the historical specimens belonged to four nominal species: Pristis zijsron (81), Anoxypristis cuspidata (39), P. pristis (30), and P. pectinata (22). An identification error of 5.41% emerged in the morphological distinction of rostra between juvenile individuals of P. pectinata and P. zijsron. The new approach of carbon and oxygen isotopes, implemented for the first time in these taxa, permitted the identification of the high-probability habitat preferences of these benthopelagic elasmobranchs in about 50% of the analysed specimens. Using this multidisciplinary approach, we successfully assigned the numerous museum rostra with lacking data to a given species and identified their candidate geographical origin, retrieving novel information and data for understanding the species distribution and ecology of past, sometimes locally/regionally extinct sawfish faunas.

Identification of a molecular signature as potential biomarker in blood of symptomatic and asymptomatic patients with carotid atherosclerosis

Background and Objectives: Carotid revascularization to prevent future vascular events is reasonable in patients with high-grade carotid stenosis. Currently, several biomarkers to predict carotid plaque development and progression have been investigated, among which microRNAs (miRs) are promising tools for the diagnosis of atherosclerosis. Methods and Results: A total of 49 participants were included in the study, divided into two main populations: Population 1 comprising symptomatic and asymptomatic inpatients, and Population 2 comprising asymptomatic outpatients. The study consisted of two main phases: a preliminary discovery phase and a validation phase, applying different techniques. MiR-profiles were performed on plasma and plaque tissue samples obtained from 4 symptomatic and 4 asymptomatic inpatients. MiRs emerging from profiling comparisons, i.e. miR-126-5p, miR-134-5p, miR-145-5p, miR-151a-5p, miR-34b, miR-451a, miR-720 and miR-1271-5p, were subjected to validation through RT-qPCR analysis in the total cohort of donors. Comparing asymptomatic and symptomatic inpatients, significant differences were reported in the expression levels of c-miRs for miR-126-5p and miR-1271-5p in blood, being more expressed in symptomatic subjects. In contrast, simultaneous evaluation of the selected miRs in plaque tissue samples did not confirm data obtained by the miR profiling, and no significant differences were observed. Using Receiver-Operating Characteristic (ROC) analysis, a circulating molecular signature (mir-126-5p, miR-1271-5p, albumin, C-reactive protein, and monocytes) was identified, allowing the distinction of the two groups in Population 1 (AUC = 0.795). Conclusions: Data emerging from this thesis suggest that c-miRs (i.e. miR-126-5p, miR-1271-5p) combined with selected haemato-biochemical parameters (albumin, C-reactive protein, and monocytes) produced a good molecular 'signature' to distinguish asymptomatic and symptomatic inpatients. C-miRs in blood do not necessarily reflect the expression levels of the same miRs in carotid plaque tissues since different mechanism can influence their expression.

Gwas analysis for the identification of molecular basis responsible for yield related traits and disease resistance in durum wheat

The PhD thesis was developed in the framework of Innovar H2020 project. This project aimed at using genomics, transcriptomics and phenotyping techniques to update varietal registration procedure used in Europe for Value of Cultivation and Use (VCU) and Distinctiness Uniformity and Stability (DUS) protocols. The phenotypic and genotypic diversity of a durum wheat panel were assessed for different agronomic traits, connected with wheat development, disease resistance and spike fertility. A panel of 253 durum wheat varieties was characterized for VCU and DUS traits and genotyped with Illumina 90K SNP Chip array (Wang et al., 2014). GWAS analysis was performed, detecting strong QTLs confirmed also by literature review. Candidate genes were identified for each trait and molecular markers will be developed to be used for marker assisted selection in breeding programs. As for disease resistance, the panel was evaluated for resistance to Soil-Borne-Cereal-Mosaic-Virus (SBCMV). A major QTL, sbm2, was detected on chromosome 2B responsible for durum wheat resistance (Maccaferri et al., 2011). The sbm2 interval was explored by fine mapping on segregant population using KASP markers and by RNASeq analysis, detecting candidate genes involved in plant-pathogen reaction. As regards yield related traits, detailed analysis was performed on the GNI-2A QTL (Milner et al., 2016), responsible for increased number spike fertility. Fine mapping analysis was performed on durum panel identifying hox2 a strong candidate gene, codifying for transcription factor protein. The gene is paralogue of GNI-1 (Sakuma et al., 2019), and it has a 4 kbp deletion responsible for increased number of florets per spikelet. To conclude, the herein reported thesis shows a complete characterization of agronomic and disease resistance traits in modern durum wheat varieties. The results obtained will augment available information for each variety, identifying informative molecular markers for breeding purposes and QTLs/candidate genes responsible for different agronomic traits.

Novel prototypes of electro-activated molecular machines

The work presented in this thesis deals with the investigation of new prototypes of molecular machines, based on rotaxane and pseudorotaxane architectures, by means of voltammetric and spectroscopic techniques. The discussion is divided in two parts. Part I concerns the investigation of electro-switchable molecular muscles, based on mechanically interlocked molecules. This study is performed on systems of increasing complexity, starting from [2]rotaxanes and arriving to polymers. In Chapters 3 and 4, [2]- and [3]rotaxanes, characterized by the presence of three stations for the macrocycle(s), are investigated. In both systems, the macrocycle(s) movement can be controlled through a combination of stimuli, resulting in a processive and directional motion. In Chapter 5, daisy chain rotaxanes, dimers of the [2]rotaxanes discussed in Chapter 3, are investigated. These systems can be switched between an extended and a contracted conformation, and they represent the monomeric units for the realization of polymeric molecular muscles. In Chapter 6, the properties of electro-switchable polymeric molecular muscles, composed by the daisy chains investigated in Chapter 5, are discussed. The repeating units of these poly-daisy chains contract and extend upon electrical stimulation, and this motion is expected to be transmitted to the polymer itself, resulting in an amplification of the effect. Part II concerns the investigation of rotaxanes and pseduorotaxanes based on heteroditopic calix[6]arenes and cationic guests. In Chapters 8 and 9, novel calix[6]arene macrocycles, functionalized with thiourea or dansyl units, and their related pseudorotaxanes are investigated. In both cases, the calix[6]arene functionalization adds new features to the pseudorotaxane. In Chapters 10 and 11, the influence of orientational isomerism on the properties of [2]- and [3]rotaxanes is investigated. The [3]rotaxanes discussed in Chapter 10 display similar properties, while the [2]rotaxanes described in Chapter 11, characterized by a calix[6]arene and a stilbazolium unit, exhibit distinct photophysical and photochemical properties.

Fluorogenic hyaluronan-based probe: characterization and use in advanced microscopy

Among all, the application of nanomaterials in biomedical research and most recently in the environmental one has opened the fields of nanomedicine and nanoremediation. Sensing methods based on fluorescence optical probe are generally requested for their selectivity, sensitivity. However, most imaging methods in literature rely on a fluorescent covalent labelling of the system. Therefore, the main aim of this project was to synthetise a biocompatible fluorogenic hyaluronan probe (HA) polymer functionalised with a rhomadine B (RB) moieties and study its behaviour as an optical probe with different materials with microscopy techniques. A derivatization of HA with RB (HA-RB) was successfully obtained providing a photophysical characterization showing a particular fluorescence mechanism of the probe. Firstly, we tested the interaction with different lab-grade micro and nanoplastics in water. Thanks to the peculiar photophysical behaviour of the probe nanoplastics can be detected with confocal microscopy and more interestingly their nature can be discriminated based on the fluorescence lifetime decay with FLIM microscopy. After, the interaction of a model plant derived metabolic enzyme GAPC1 undergoing oxidative-triggered aggregation was explored with the HA-RB. We highlighted the probe interaction with the protein even at early stage of the kinetic. Moreover, nanoparticle tracking analysis (NTA) experiment demonstrates that the probe is in fact able to interact with the small pre-aggregates in the early stage of the aggregation kinetic. Ultimately, we focused on the possibility to apply the probe in a super resolution microscopy technique, PALM, exploiting its aspecific interaction to characterize the surface topography of PTFE polydisperse microplastics. Optimal conditions were reached at high concentration of the probe (70 nM) where 0.5-5 nM is always advisable for this technique. Thanks to the polymeric nature and fluorescence mechanism of the probe, this technique was able to reveal features of PTFE surface under the diffraction limit (< 250 nm).

Exploiting molecular simulations as an atomic-level microscope to elucidate clinically relevant cation chloride cotransporters

The cation chloride cotransporters (CCCs) represent a vital family of ion transporters, with several members implicated in significant neurological disorders. Specifically, conditions such as cerebrospinal fluid accumulation, epilepsy, Down’s syndrome, Asperger’s syndrome, and certain cancers have been attributed to various CCCs. This thesis delves into these pharmacological targets using advanced computational methodologies. I primarily employed GPU-accelerated all-atom molecular dynamics simulations, deep learning-based collective variables, enhanced sampling methods, and custom Python scripts for comprehensive simulation analyses. Our research predominantly centered on KCC1 and NKCC1 transporters. For KCC1, I examined its equilibrium dynamics in the presence/absence of an inhibitor and assessed the functional implications of different ion loading states. In contrast, our work on NKCC1 revealed its unique alternating access mechanism, termed the rocking-bundle mechanism. I identified a previously unobserved occluded state and demonstrated the transporter's potential for water permeability under specific conditions. Furthermore, I confirmed the actual water flow through its permeable states. In essence, this thesis leverages cutting-edge computational techniques to deepen our understanding of the CCCs, a family of ion transporters with profound clinical significance.