Nanoscale surface properties and interaction with fundamental biomolecules of chlorite and phlogopite

The surface properties of minerals have important implications in geology, environment, industry and biotechnology and for certain aspects in the research on the origin of life. This research project aims to widen the knowledge on the nanoscale surface properties of chlorite and phlogopite by means of advanced methodologies, and also to investigate the interaction of fundamental biomolecules, such as nucleotides, RNA, DNA and amino acid glycine with the surface of the selected phyllosilicates. Multiple advanced and complex experimental approaches based on scanning probe microscopy and spatially resolved spectroscopy were used and in some cases specifically developed. The results demonstrate that chlorite exposes at the surface atomically flat terraces with 0.5 nm steps typically generated by the fragmentation of the octahedral sheet of the interlayer (brucitic-type). This fragmentation at the nanoscale generates a high anisotropy and inhomogeneity with surface type and isomorphous cationic substitutions determining variations of the effective surface potential difference, ranging between 50-100 mV and 400-500 mV, when measured in air, between the TOT surface and the interlayer brucitic sheet. The surface potential was ascribed to be the driving force of the observed high affinity of the surface with the fundamental biomolecules, like single molecules of nucleotides, DNA, RNA and amino acids. Phlogopite was also observed to present an extended atomically flat surface, featuring negative surface potential values of some hundreds of millivolts and no significant local variations. Phlogopite surface was sometimes observed to present curvature features that may be ascribed to local substitutions of the interlayer cations or the presence of a crystal lattice mismatch or structural defects, such as stacking faults or dislocation loops. Surface chemistry was found similar to the bulk. The study of the interaction with nucleotides and glycine revealed a lower affinity with respect to the brucite-like surface of chlorite.

Data management and data analysis in the large European projects GEHA (GEnetics of Healthy Aging) and NU-AGE (NUtrition and AGEing): a bioinformatic approach

The aging process is characterized by the progressive fitness decline experienced at all the levels of physiological organization, from single molecules up to the whole organism. Studies confirmed inflammaging, a chronic low-level inflammation, as a deeply intertwined partner of the aging process, which may provide the “common soil” upon which age-related diseases develop and flourish. Thus, albeit inflammation per se represents a physiological process, it can rapidly become detrimental if it goes out of control causing an excess of local and systemic inflammatory response, a striking risk factor for the elderly population. Developing interventions to counteract the establishment of this state is thus a top priority. Diet, among other factors, represents a good candidate to regulate inflammation. Building on top of this consideration, the EU project NU-AGE is now trying to assess if a Mediterranean diet, fortified for the elderly population needs, may help in modulating inflammaging. To do so, NU-AGE enrolled a total of 1250 subjects, half of which followed a 1-year long diet, and characterized them by mean of the most advanced –omics and non –omics analyses. The aim of this thesis was the development of a solid data management pipeline able to efficiently cope with the results of these assays, which are now flowing inside a centralized database, ready to be used to test the most disparate scientific hypotheses. At the same time, the work hereby described encompasses the data analysis of the GEHA project, which was focused on identifying the genetic determinants of longevity, with a particular focus on developing and applying a method for detecting epistatic interactions in human mtDNA. Eventually, in an effort to propel the adoption of NGS technologies in everyday pipeline, we developed a NGS variant calling pipeline devoted to solve all the sequencing-related issues of the mtDNA.

Transcriptional functions of DNA Topoisomerases at a genome-wide scale and a single gene levels

The DNA topology is an important modifier of DNA functions. Torsional stress is generated when right handed DNA is either over- or underwound, producing structural deformations which drive or are driven by processes such as replication, transcription, recombination and repair. DNA topoisomerases are molecular machines that regulate the topological state of the DNA in the cell. These enzymes accomplish this task by either passing one strand of the DNA through a break in the opposing strand or by passing a region of the duplex from the same or a different molecule through a double-stranded cut generated in the DNA. Because of their ability to cut one or two strands of DNA they are also target for some of the most successful anticancer drugs used in standard combination therapies of human cancers. An effective anticancer drug is Camptothecin (CPT) that specifically targets DNA topoisomerase 1 (TOP 1). The research project of the present thesis has been focused on the role of human TOP 1 during transcription and on the transcriptional consequences associated with TOP 1 inhibition by CPT in human cell lines. Previous findings demonstrate that TOP 1 inhibition by CPT perturbs RNA polymerase (RNAP II) density at promoters and along transcribed genes suggesting an involvement of TOP 1 in RNAP II promoter proximal pausing site. Within the transcription cycle, promoter pausing is a fundamental step the importance of which has been well established as a means of coupling elongation to RNA maturation. By measuring nascent RNA transcripts bound to chromatin, we demonstrated that TOP 1 inhibition by CPT can enhance RNAP II escape from promoter proximal pausing site of the human Hypoxia Inducible Factor 1 (HIF-1) and c-MYC genes in a dose dependent manner. This effect is dependent from Cdk7/Cdk9 activities since it can be reversed by the kinases inhibitor DRB. Since CPT affects RNAP II by promoting the hyperphosphorylation of its Rpb1 subunit the findings suggest that TOP 1inhibition by CPT may increase the activity of Cdks which in turn phosphorylate the Rpb1 subunit of RNAP II enhancing its escape from pausing. Interestingly, the transcriptional consequences of CPT induced topological stress are wider than expected. CPT increased co-transcriptional splicing of exon1 and 2 and markedly affected alternative splicing at exon 11. Surprisingly despite its well-established transcription inhibitory activity, CPT can trigger the production of a novel long RNA (5’aHIF-1) antisense to the human HIF-1 mRNA and a known antisense RNA at the 3’ end of the gene, while decreasing mRNA levels. The effects require TOP 1 and are independent from CPT induced DNA damage. Thus, when the supercoiling imbalance promoted by CPT occurs at promoter, it may trigger deregulation of the RNAP II pausing, increased chromatin accessibility and activation/derepression of antisense transcripts in a Cdks dependent manner. A changed balance of antisense transcripts and mRNAs may regulate the activity of HIF-1 and contribute to the control of tumor progression After focusing our TOP 1 investigations at a single gene level, we have extended the study to the whole genome by developing the “Topo-Seq” approach which generates a map of genome-wide distribution of sites of TOP 1 activity sites in human cells. The preliminary data revealed that TOP 1 preferentially localizes at intragenic regions and in particular at 5’ and 3’ ends of genes. Surprisingly upon TOP 1 downregulation, which impairs protein expression by 80%, TOP 1 molecules are mostly localized around 3’ ends of genes, thus suggesting that its activity is essential at these regions and can be compensate at 5’ ends. The developed procedure is a pioneer tool for the detection of TOP 1 cleavage sites across the genome and can open the way to further investigations of the enzyme roles in different nuclear processes.

A hybrid technology for parallel recording of single ion channels

Hybrid technologies, thanks to the convergence of integrated microelectronic devices and new class of microfluidic structures could open new perspectives to the way how nanoscale events are discovered, monitored and controlled. The key point of this thesis is to evaluate the impact of such an approach into applications of ion-channel High Throughput Screening (HTS)platforms. This approach offers promising opportunities for the development of new classes of sensitive, reliable and cheap sensors. There are numerous advantages of embedding microelectronic readout structures strictly coupled to sensing elements. On the one hand the signal-to-noise-ratio is increased as a result of scaling. On the other, the readout miniaturization allows organization of sensors into arrays, increasing the capability of the platform in terms of number of acquired data, as required in the HTS approach, to improve sensing accuracy and reliabiity. However, accurate interface design is required to establish efficient communication between ionic-based and electronic-based signals. The work made in this thesis will show a first example of a complete parallel readout system with single ion channel resolution, using a compact and scalable hybrid architecture suitable to be interfaced to large array of sensors, ensuring simultaneous signal recording and smart control of the signal-to-noise ratio and bandwidth trade off. More specifically, an array of microfluidic polymer structures, hosting artificial lipid bilayers blocks where single ion channel pores are embededed, is coupled with an array of ultra-low noise current amplifiers for signal amplification and data processing. As demonstrating working example, the platform was used to acquire ultra small currents derived by single non-covalent molecular binding between alpha-hemolysin pores and beta-cyclodextrin molecules in artificial lipid membranes.

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.

Innovative Strategies for the Synthesis of Biologically Active Small Molecules

The post genomic era, set the challenge to develop drugs that target an ever-growing list of proteins associated with diseases. However, an increase in the number of drugs approved every year is nowadays still not observed. To overcome this gap, innovative approaches should be applied in drug discovery for target validation, and at the same time organic synthetic chemistry has to find new fruitful strategies to obtain biologically active small molecules not only as therapeutic agents, but also as diagnostic tools to identify possible cellular targets. In this context, in view of the multifactorial mechanistic nature of cancer, new chimeric molecules, which can be either antitumor lead candidates, or valuable chemical tools to study molecular pathways in cancer cells, were developed using a multitarget-directed drug design strategy. According to this approach, the desired hybrid compounds were obtained by combining in a single chemical entity SAHA analogues, targeting histone deacetylases (HDACs), with substituted stilbene or terphenyl derivatives able to block cell cycle, to induce apoptosis and cell differentiation and with Sorafenib derivative, a multikinase inhibitor. The new chimeric derivatives were characterized with respect to their cytotoxic activity and their effects on cell cycle progression on leukemia Bcr-Abl-expressing K562 cell lines, as well as their HDACs inhibition. Preliminary results confirmed that one of the hybrid compounds has the desired chimeric profile. A distinct project was developed in the laboratory of Dr Spring, regarding the synthesis of a diversity-oriented synthesis (DOS) library of macrocyclic peptidomimetics. From a biological point of view, this class of molecules is extremely interesting but underrepresented in drug discovery due to the poor synthetic accessibility. Therefore it represents a valid challenge for DOS to take on. A build/couple/pair (B/C/P) approach provided, in an efficient manner and in few steps, the structural diversity and complexity required for such compounds.

Elemetary processes of radiation damage in organic molecules of biological interest

It was observed in the ‘80s that the radiation damage on biological systems strongly depends on processes occurring at the microscopic level, involving the elementary constituents of biological cells. Since then, lot of attention has been paid to study elementary processes of photo- and ion-chemistry of isolated organic molecule of biological interest. This work fits in this framework and aims to study the radiation damage mechanisms induced by different types of radiations on simple halogenated biomolecules used as radiosensitizers in radiotherapy. The research is focused on the photofragmentation of halogenated pyrimidine molecules (5Br-pyrimidine, 2Br-pyrimidine and 2Cl-pyrimidine) in the VUV range and on the 12C4+ ion-impact fragmentation of the 5Br-uracil and its homogeneous and hydrated clusters. Although halogen substituted pyrimidines have similar structure to the pyrimidine molecule, their photodissociation dynamics is quite different. These targets have been chosen with the purpose of investigating the effect of the specific halogen atom and site of halogenation on the fragmentation dynamics. Theoretical and experimental studies have highlighted that the site of halogenation and the type of halogen atom, lead either to the preferential breaking of the pyrimidinic ring or to the release of halogen/hydrogen radicals. The two processes can subsequently trigger different mechanisms of biological damage. To understand the effect of the environment on the fragmentation dynamic of the single molecule, the ion-induced fragmentation of homogenous and hydrated clusters of 5Br-uracil have been studied and compared to similar studies on the isolated molecule. The results show that the “protective effect” of the environment on the single molecule hold in the homogeneous clusters, but not in the hydrated clusters, where several hydrated fragments have been observed. This indicates that the presence of water molecules can inhibit some fragmentation channels and promote the keto-enol tautomerization, which is very important in the mutagenesis of the DNA.

Electrochemical surface modification of Single walled carbon nanotubes and graphene-based electrodes for (bio)sensing applications

Sensors are devices that have shown widespread use, from the detection of gas molecules to the tracking of chemical signals in biological cells. Single walled carbon nanotube (SWCNT) and graphene based electrodes have demonstrated to be an excellent material for the development of electrochemical biosensors as they display remarkable electronic properties and the ability to act as individual nanoelectrodes, display an excellent low-dimensional charge carrier transport, and promote surface electrocatalysis. The present work aims at the preparation and investigation of electrochemically modified SWCNT and graphene-based electrodes for applications in the field of biosensors. We initially studied SWCNT films and focused on their topography and surface composition, electrical and optical properties. Parallel to SWCNTs, graphene films were investigated. Higher resistance values were obtained in comparison with nanotubes films. The electrochemical surface modification of both electrodes was investigated following two routes (i) the electrografting of aryl diazonium salts, and (ii) the electrophylic addition of 1, 3-benzodithiolylium tetrafluoroborate (BDYT). Both the qualitative and quantitative characteristics of the modified electrode surfaces were studied such as the degree of functionalization and their surface composition. The combination of Raman, X-ray photoelectron spectroscopy, atomic force microscopy, electrochemistry and other techniques, has demonstrated that selected precursors could be covalently anchored to the nanotubes and graphene-based electrode surfaces through novel carbon-carbon formation.

Design and Synthesis of Naphthalene Diimides Based Small Molecules as Anticancer Agents: Targeting the Polyamine Transporter, G-quadruplex Structures and HDAC

Cancer is a multifactorial disease characterized by a very complex etiology. Basing on its complex nature, a promising therapeutic strategy could be based by the “Multi-Target-Directed Ligand” (MTDL) approach, based on the assumption that a single molecule could hit several targets responsible for the pathology. Several agents acting on DNA are clinically used, but the severe deriving side effects limit their therapeutic application. G-quadruplex structures are DNA secondary structures located in key zones of human genome; targeting quadruplex structures could allow obtaining an anticancer therapy more free from side effects. In the last years it has been proved that epigenetic modulation can control the expression of human genes, playing a crucial role in carcinogenesis and, in particular, an abnormal expression of histone deacetylase enzymes are related to tumor onset and progression. This thesis deals with the design and synthesis of new naphthalene diimide (NDI) derivatives endowed with anticancer activity, interacting with DNA together with other targets implicated in cancer development, such as HDACs. NDI-polyamine and NDI-polyamine-hydroxamic acid conjugates have been designed with the aim to provide potential MTDLs, in order to create molecules able simultaneously to interact with different targets involved in this pathology, specifically the G-quadruplex structures and HDAC, and to exploit the polyamine transport system to get selectively into cancer cells. Macrocyclic NDIs have been designed with the aim to improve the quadruplex targeting profile of the disubstituted NDIs. These compounds proved the ability to induce a high and selective stabilization of the quadruplex structures, together with cytotoxic activities in the micromolar range. Finally, trisubstituted NDIs have been developed as G-quadruplex-binders, potentially effective against pancreatic adenocarcinoma. In conclusion, all these studies may represent a promising starting point for the development of new interesting molecules useful for the treatment of cancer, underlining the versatility of the NDI scaffold.