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Meccanismi cellulari implicati nell'effetto antitumorale del cannabidiolo su cellule di glioma umano U87-MG e caratterizzazione delle sue proprietà antiangiogeniche.
No full textIn this study we evaluated the ability of the non-psychoactive cannabinoid compound cannabidiol (CBD) to limit glioma cells invasiveness and to interfere with the most relevant intracellular pathways involved in tumor development, together with its ability to modulate angiogenic process.
We found that CBD inhibited U87-MG cells invasiveness, caused a down-regulation of ERK and Akt activation, reduced HIF-1α expression and decreased the level of several proteins specifically involved in growth, invasion and angiogenesis.
CBD also induced endothelial cell cytostasis, without inducing apoptosis, inhibited their migration, invasion and sprouting in vitro, and angiogenesis in vivo, with correspondent down-modulation of several angiogenesis-related molecules.
These results provide new insight into CBD antitumor action, showing multiple tumoral features and molecular pathways affected by CBD. Moreover, its dual effect on both glioma and endothelial cells reinforces the hypothesis of its possible use as effective anticancer agent.
Finally we evaluated the antitumor effects of other non-psychoactive cannabinoid compounds, not thoroughly investigate so far.
These compounds inhibited, with different extents, glioma cell viability, migration and invasiveness, through mechanisms nor cannabinoid or vanilloid receptors-mediated. Our results contribute to clarify the pharmacology of these molecules, although further studies are needed to understand cellular mechanisms involved in their effects
Identification and characterization of small non-coding RNA molecules transcribed in Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae) larvae parasitized by Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae).
No full textToxoneuron nigriceps (Hymenoptera, Braconidae) is an endophagous parasitoid of the tobacco budworm, Heliothis virescens (Lepidoptera, Noctuidae). Parasitized larvae show a complex array of pathological symptoms including immune response suppression, largely due to a symbiotic virus, named TnBV, belonging to the family Polydnaviridae (PDV). A striking characteristic of the TnBV, as well as the other PDV genomes sequenced to date, is the large preponderance of non-coding regions, which raises the question of their functional relevance. To start addressing this question, we decided to analyze the small RNA fraction present in haemocytes of parasitized larvae and search for TnBV-derived sequences that might interfere with the host immune response.
To investigate this hypothesis in the case of TnBV, a specialized cDNA library, representative of the small RNA fraction present in haemocytes of parasitized larvae, was prepared. The generated cDNA library was validated by two types of quality control analyses.
Real-time PCR Absolute Quantification was performed using a standard curve method. This analysis essentially allowed me to evaluate the concentration of the library and also to obtain indications on its purity, by assessing the presence and relative abundance of adapter dimers contaminants.
A further quality test was carried out by cloning a small amount of the cDNA products in a plasmid vector and by sequencing through conventional technology few randomly selected clones. Two cloned sequences turned to be microRNA species already known in other organisms, miR-184 and miR-970, evolutionarily conserved from insects to mammals. In Drosophila melanogaster miR-184 is expressed in all developmental stages (Ping Li et al., 2011). Moreover, functional analysis identified a role for this miRNA in the female germline, where it is involved in multiple steps of oogenesis (Iovino et al., 2009).
The presence of authentic miRNA species in our library, as well as the finding of a correct size range (21-27) in our cloned cDNA sequences represented valid quality control criteria, empowering further analysis of the cDNA library by high-throughput sequencing technologies.
Specifically, cDNA sequencing was performed using the Illumina platform and resulted in about 6 million total reads. Sequence alignment with the TnBV genome was then performed using the FAST (Fast Aligning of Short Tags) software developed by the Institute of Applied Genomics of Udine. These preliminary analyses allowed the identification of 52 cDNA sequences, ranging in size between 20 and 27 nucleotides, that align with the viral genome. 46 are present in the viral genome in single copy, while 6 are located in two different positions and are therefore represented twice. These sequences were extensively analyzed by bioinformatical tools, in particular computational methods based on secondary structure prediction to assess the ability of these genomic regions to generate appropriate small RNA precursor molecules. Specific analyses were performed using a web tool, MirEval. 5 sequences were selected as potential miRNA species, deriving from viral genomic regions that show structural features typical of precursor-miRNA (pre-miRNA) molecules. RT-PCR experiments of these genomic regions, aimed at detecting presumptive primary-miRNA (pri-miRNA) species, indicated that these genomic regions are actively transcribed in haemocytes, 6 and 12 hours after parasitization. In parallel, transcriptional profiling of the TnBV genomic regions where the remaining presumptive TnBV small ncRNA sequences were mapped, as well as of randomly selected regions, that do not contain any protein coding genes or any putative small ncRNAs, indicated that the non-coding portion of the TnBV genome is extensively transcribed in host haemocytes. These analyses indicated that most, if not all, of the non-coding portion of the TnBV genome is transcribed in host haemocytes. This unexpected finding raises several questions concerning: 1) the functional meaning of generalized transcription and 2) the possible existance of specific mechanisms acting downstream of transcription to discriminate biologically relevant RNA molecules from “transcriptional noise”.
Functional characterization of the sequences identified as potential microRNAs is expected to shed light on the role played by the non-coding regions of the TnBV genome during parasitization. Furthermore, future work aimed at target identification may disclose the possible involvement of these sequences in the alteration of host immune system. The generated information will be essential for defining a more comprehensive model of the immunosuppression phenomena occurring during parasitization
Mechanisms involved in the adsorption of bioinsecticides and strategies to enhance their passage through the insect midgut.
No full textDuring the past decade a number of products have emerged from the effort to develop alternative biopesticidal technologies. The most recent approach to integrated pest management is based on the detection of new genes encoding for polypeptides with potential insecticidal activity, with a particular attention to biopesticides derived from viruses, microorganisms, fungi, plants and insects. Bacteria and viruses themselves can be used for biological control purpose. In most cases, biopesticides are molecules that have haemocoelic targets and must pass undegraded the gut barrier in order to exert their activity. Therefore for an effective oral delivery of these molecules it will be crucial to develop basic information on the molecular mechanisms mediating the absorption of proteins and peptides by the insect gut and to develop strategies to facilitate their passage through the midgut barrier. These issues have been the subject of my research project during these three years. In particular, I characterized the strategies adopted by Junonia coenia densovirus (JcDNV) to cross the midgut barrier of the lepidopteran pest Spodoptera frugiperda. I also studied a possible mechanism to increase the protein uptake in insect midgut characterizing the mechanisms involved in the internalization of selected Cell Penetrating Peptides (Tat, Penetratin, L-oligoarginine and D-oligoarginine) through the plasma membrane of lepidopteran larveae midgut cells. Even though these two aspects represent the main part of my PhD project, I have also collaborated to the study devoted to the identification of strategies to increase the permeability of the paracellular route
DSDM (Digital Slide Dynamic Morphometry): targeting tumor budding in colorectal cancer.
No full textBackground: In histopathology, quantitative assessment of various morphologic features is based on published methods that were conceived on specific areas observed through the microscope and microscopical objective used. Failure to reproduce the same reference field size if using a different microscope may change the score assessed. When visualizing a digital slide on a computer screen, through a dedicated viewer, it is possible to select the adequate objective-magnification (zoom toolbar). However, the field of view is rectangular, different from the circular field area viewed by optical microscopy. In addition, the evaluation of the size of the selected area of the digital slide is not immediately evident and must be estimated with the draw functions of the viewer or the scale/axes grid.
Methods: Using the tools offered by ImageScope, the virtual slide viewer of Aperio Technologies (Vista, USA), a system was conceived to reproduce the various methods published for assessing tumor budding, an important prognostic factor in colorectal cancer. Each method, based on counting tumor budding within a specific area, was reproduced as a colored circle, corresponding to a graphic overlay layer. The various circles were grouped in a target-like shape and then exported and saved as an .xml file. A web page was created giving access to 100 whole-slide digital scans. When each digital slide was opened the file containing the target-like area had to be imported in order to move it onto a tumor budding “hot spot” and to perform the
measurements. Eighteen international experts in gastrointestinal pathology were invited to participate.
Results: Twelve investigators completed the task, the majority of them performing the multiple assessments of each case in less than 12 minutes.
Conclusions: The assessing system proposed appeared to be feasible for pathologists. It allows precise application of methods originally conceived for optical microscopy. The graphical layers that make the tool are freely moved within the digital slide: this system was named DSDM (Digital Slide Dynamic Morphometry). This telepathology extension has various potential applications and might be a useful tool for histopathological assessment of diagnostic parameters that need to be quantified
The X-ray behaviour of Fermi/LAT pulsars.
No full textMore than 40 years after the discovery of Isolated Neutron Stars, the comprehension of their physics is still rather poor. This thesis is based on a program of multiwavelength observations of pulsars which yielded new and important pieces of information about the overall proprieties of this class of sources.
The thesis is organized as follows:
- In chapter 1 we give a very brief overview of the current status of the under-standing of Isolated Neutron Stars. We also talk about the Fermi revolution that occurred in the last three years, focusing on the Fermi contribution to the knowledge of neutron stars. Then, we describe the results led by the synergy between X-ray and °-ray bands.
- In chapter 2 we give a detailed description of X-ray analyses we did and surprising results we obtained for two different radio-quiet pulsars. Such neutron stars, J0007+7303 and J0357+3205, can be considered ”extreme” in the Fermi pulsars’ zoo due to their energetics and ages. Both the X-ray observations and analyses are very different so that they can be considered as the standing-up examples of all the following analyses.
- In chapter 3 we describe the analysis we done in the X-ray band and briefly report the obtained spectra of each pulsar and its nebula, if present. Then, we study the X-ray and °-ray pulsars’ luminosities as a function of their rotational energies and ages in order to find any relationship between these values and any difference between the two populations of radio-quiet and radio-loud pulsars.
- In chapter 4 we report the ”identity card” of all Fermi pulsars, the detailed description of the analyses done and results obtained for each pulsar.
Finally, in appendix we report our accepted proposals of the most significative X-ray observations used in this thesis plus the article on the X-ray behaviour of Fermi/LAT pulsars we published on the Astrophyisical Journal
Cycloisomerization reactions catalyzed by transition metal complexes. Synthesis of oxygen- and nitrogen- containing heterocyclic compounds.
No full textThe increasing need for environmentally responsible means of preparing a wide diversity of chemical products demanded by society drives the quest for synthetic efficiency. Transition metal-catalyzed cyclizations of polyunsaturated system, which are both selective and atomeconomical, represent an important starting point for this long-term goal.
We describe herein four different class of transition metal-catalyzed reactions that provided an atom- and step-economical entry into a range of oxygen- and nitrogen-containing heterocycles: (1) ring-closing metathesis, (2) Pauson-Khand reactions, (3) platinum-, goldand iridium-catalyzed electrophilic activation of alkynes and (4) gold-catalyzed cyclizations of allenes. All these transformations allow for a rapid increase in molecular complexity from relatively simple starting materials. Moreover, since these processes exhibit an excellent chemoselectivity towards C-C ! systems, the cyclized products usually retain different functional groups that can be used for further synthetic transformations.
Manuscripts adding to the scope of transition metal-catalyzed reactions are being published at a rapid rate attesting to their interest in the chemical community. With the development of new asymmetric methodologies, the transformations described in this thesis will continue to occupy, in the next future, a remarkable role in organic synthesis
Kripke semantics and tableau procedures for constructive description logics.
No full textIn this work we present the decidable constructive description logic KALC: the logic is based on a Kripke-style semantics for the language of the description logic ALC and it is directly inspired by the Kripke semantics for first order intuitionistic logic. We study the constructive properties of this logic and its relations with classical semantics. Then, by means of an example, we show how its semantics is suitable for the description of incomplete and dynamic knowledge. We then introduce a tableau calculus for this logic and we prove its completeness with respect to KALC semantics. Most notably, by proving the completeness and termination results for such calculus, we obtain an effective proof search algorithm for our logic. We also study the relations of KALC with our previous proposals for constructive description logics, with first order intuitionistic logic and with well-known intuitionistic multi-modal logics. We conclude by presenting an application for a different constructive semantics for KALC in the context of Semantic Web services compositio
