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Analisi mutazionale dei geni RET e RAS in un’ampia casistica di carcinomi midollari della tiroide
No full textEnergetics of Cerebral Cortex: Metabolic Modeling and In Vivo NMR
No full textThe energy metabolism of cerebral cortex adapts to the increase in metabolic demand which occurs during enhanced cortical activity (e.g. sensory stimulation). This contributes to the generation of the imaging and spectroscopic signals that are utilized to monitor and appraise the function of the brain in situ. Importantly, the simple paradigm of “neuronal activity” is insufficient to interpret experimental outcomes, as what is observed depends on the balance between (i) activity patterns, whether excitatory or inhibitory; (ii) information processing mechanisms, specifically input/output or synaptic/spiking activity; (iii) stimulation of cell-specific (primarily neuronal and astrocytic) functional and metabolic pathways. Understanding the basis of the metabolic response of the cortical tissue to stimulation (i.e. neurometabolic coupling) is an important goal for neurosciences.
In the present work, I studied the coupling between activity and energy metabolism of the human cerebral cortex by combining theoretical and experimental approaches. First, I adapted and used the information about the energy consumed by cortical signaling processes in order to develop a kinetic model of carbohydrate metabolism (Chapters 1 and 2). By encompassing the current knowledge about the regulation of energy supply and demand in cortical tissue, the model predicted that the metabolic response of the cerebral cortex might be strongly dependent on the balance between spiking and synaptic activity. Then, I designed an experiment for the measurement of vascular and metabolic response of the primary visual cortex to specific visual stimulations (Chapter 3). In particular, by acting on the temporal frequency of achromatic luminance and isoluminant chromatic flickering visual stimulations, I aimed at altering the balance between local input processing (synaptic activity) and output firing (spiking activity) through changes in intracortical inhibition.
The modeling and experimental results support the hypothesis (discussed in Chapter 4) that spiking activity, which is identified by axonal action potentials propagation, hinges on fast metabolic pathways that do not depend on oxygen (i.e. anaerobic). This suggests that the up-regulation of anaerobic metabolism during enhanced cortical activity is primarily due to the long-range communication between different areas of the cerebral cortex, more than to local processing within an individual cortical area. Identifying the potential significance of this mechanism is the subject of my future research
Design, Synthesis and anti-HIV Activity of New Indolyl Aryl Sulfones (IASs): Structure-Activity Relationship of Substituents on the 2-Carboxyamides Function
No full textSINTHESES AND FUNCTIONALIZATION OF POLY(L-LACTIDE) BASED NANOCOMPOSITES SYSTEMS FOR SELECTIVE AND CONTROLLED DRUG RELEASE IN BIOMEDICAL USES
No full textIn this thesis, the research activity, focused on two investigations topics, both addressed to the preparation and characterization of poly(L-lactide) (PLLA) based nano-materials for biomedical applications, is reported. In particular, the research deals with the preparation of a new polymeric substrate for vaccine (line 1) and superparamagnetic nanoparticles-PLLA core-shell nanocomposites for targeted and controlled release of anti-tumor drugs (line 2).The polymer we used in both the researches, the poly (L-lactide) (PLLA), is one of the most investigated synthetic product in the biomedical field because of its good mechanical properties, biocompatibility and biodegradability.
In this work the PLLA has been used in:
- Line 1, as a possible biodegradable carrier for the development of a vaccine. We employed the polymer in guise of lamellar single crystals (PLLAsc) growth from dilute solution, characterized by a 1D-nano-2D-micro morphology and by a high specific area.
- Line 2, as the outer coating of micro-nano-superparamagnetic particles (MNPs) for the realization of nano-composites with core-shell morphology to be used for the preparation of drug-delivery systems.
Line 1. In the last years, the research on innovative immunization systems took into account the use of polymer micro or nano particles as substrate, vehicle or/and adjuvant for development of new vaccines.
In traditional vaccination systems, the conventional antigens are highly immunogenic and induce a strong and durable protective response in the individual immune system. At the same time, the antigen may be potentially harmful or induce dangerous side effects. In contrast, vaccines based on antigens composed of sub-molecular units, obtained by recombinant DNA techniques, usually has negligible side effects. Unfortunately, with rare exceptions, this type of antigen induces low antibody responses and need an "adjuvant" that can enhance the immunogenicity. The research in this field is boosted by the possibility to use immunotherapy for the tumors prevention and eradication. In fact, the cancer, during the malignant progression, is able to evade the body immune response by developing mechanisms that hide its presence to the host. If the immune system is activated by a proper vaccine against these tumours, the body could be able to kill the tumoral cells
In this research, the E7 protein of human papillomavirus (HPV16), related to the formation of cervical cancer, has been used for the preparation of a polymer/antigen vaccine. PLLA single crystals (PLLAsc), characterized by an lamellar hexagonal shape with lateral dimensions of about 15 micron and thickness of 10 nm, were used as biodegradable antigene carrier.
To increase the hydrophilicity and provide the PLLAsc of active sites useful to the subsequent protein adsorption, a controlled reaction of aminolysis with tetraethylenepentamine (TEPA) was carried out on the surface ester bond of PLLAsc (APLLAsc).The pristine and functionalized single crystals were characterized and the effectiveness of the APLLAsc-E7 vaccine was evaluated by tumour protection experiments on mice. It has been observed that, unlike the behaviour of non-functionalized single crystals, the lamellae have a strong adjuvant effect. The results opens interesting perspectives for a possible use of the APLLAsc-E7 system as a single dose vaccine.
Line 2. Superparamagnetic nano-particles (MNPs) represent the object of an extensive research in the material science and medicine fields. In fact, by a proper selection of the composition, it is possible to prepare hybrid inorganic core-polymer shell nanocomposites able to fulfill diagnostic and therapeutic tasks.
The MNPs are magnetized only in presence of an external magnetic fields and show no residual magnetism when the field is removed, that is no hysteresis in the magnetization curve is observed. In this class of nanoparticles are included superparamagnetic iron oxides and iron oxides doped with +2 cations (M: Mn, Co, Fe, Ni) to form MFe2O4 spinel structures.
The coating of MNPs by organic shell is mandatory to overcome their agglomeration driven by their high surface-to-volume ratio. Moreover, the outermost sheath can increase the nanoparticles circulation time in vivo before their clearance by macrophages in the reticulo-endothelial system.
The MNPs can be directed toward a target organ or tissue by external magnetic field. Once they reach the target they can perform their therapeutic task by means physical or chemical process. As far as the former is concerned, when subject to alternating external magnetic field, magnetic nanoparticles absorb energy and convert it into heat through a mechanism called magnetic hyperthermia. The generated local temperature increase, up to 41-47 °C, can kills the surrounding tissue cells, such as cancer cells. As far the chemical action, MNPs can be loaded with a drug and used as drug delivery vehicles. The possibility to direct them on a specific target has the advantage to increase the potency of many therapeutics and reduce their possible systemic toxicity. Moreover, the drug release can be trigged by external stimuli. The local magnetic hyperthermia, for instance, can activate polymer transition, like volume collapse of a thermo-sensitive polymer shell, and induce an accelerative drug release.
In this research, superparamagnetic nanoparticles (MNPs), composed of manganese and iron oxides (MnFe2O4) and synthesized by the technique of water-in-oil microemulsion, have been used.The obtained MNPs have sizes in the order of 10 nm and present superparamagnetic behaviour. Three different methods were used to coat the nanoparticles with PLLA and to obtain a core-shell nanocomposite:
- Grafting from: -OH groups on the surface of nanoparticles have been used as initiators for catalyzed ring opening polymerization (ROP) of L-lactide.
- Grafting to: a series of carboxylated-poly(L-lactide) with different molecular weight (CPLLAs), based on 2,2-bis(hydroxymethyl) propionic acid (DMPA) as initiator, have been prepared by ROP. Then, CPLLAs were adsorbed onto nanoparticles surface by exploiting the specific interaction of the carboxylic acid with the MNPs surface.
- One step: the monomer (L-lactide), the initiator (DMPA), MNPs and the catalyst were reacted simultaneously.
All the CPLLAs and the three types of nano-composites were chemical and physico-chemical characterized. Even if the grafting from and one step methods are more attractive from the scale-up point of view, they have provided low yields. The grafting to method was observed to be more controllable in terms of molecular weights and thickness of the shell. The nanocomposites were then loaded with an anti-tumor model drug (Usnic acid) and the static and dynamic release of the drug in PBS was evaluated
DETECTING MUTUALLY EXCLUSIVE INTERACTIONS IN PROTEIN-PROTEIN INTERACTION MAPS
No full textProteins are responsible for an impressive large variety of functions. To properly understand the significance of protein-protein interactions in the cell it is important to address two problems: first, is identification of the different interactions that are involved in each biological function, and, second, is determining how proteins interact and the consequences of the interaction.
The identification of protein interactions by high-throughput experiments has led to the development of a number of methods for their analysis, producing, in the last years, a vast amount of interacting data. However, there are at least two issues that arise from the analysis of such experimental maps, these are, on one side, the significant number of false positives they contain and, on the other, the difficulty in distinguishing whether, when more than one protein interact with the same partner, they can do so simultaneously, i.e. whether their interaction is mutually exclusive.
The general strategy we describe is based on the combination of known three-dimensional structures with protein-protein interaction networks to determine which of the multiple interactions or connections that are made by a hub can occur in mutually exclusive fashion, and, in such cases, identify, whenever is possible, the shared similarities in their binding regions, concluding that their interaction has to be mutually exclusive (i.e. not simultaneous) and that the region identified by similarity is indeed the interaction site.
We applied this strategy to the interactomes of seven organisms. We show that our methodology allows the identification of mutually exclusive interactions with accuracy higher than 77%. The procedure also allows us to predict which residues are likely to be in the binding interface of the nodes, and in a significant number of cases (between 63% and 75%) we correctly identify at least one of them (5 on average) and this has obvious implications for helping to reduce the search space in docking procedures. The coverage of the method varies substantially for different organisms, as it could be expected, however it does reach 42% for human and more than 36% for yeast averaging at about 25%. These figures are bound to increase with time both thanks to the progress in experimental methods and, possibly, to the increasing reliability of modeling techniques. For this reason, we also introduce here the Estrella server that embodies this strategy, is designed for users interested in validating specific hypotheses about the functional role of a protein-protein interaction and it also allows access to pre-computed data for seven organisms.King Abdullah University of Science and Technology (KAUST
Resource for benchmarking the applicability of protein structure models
No full textThe function of a protein is closely related to the structure it attains. The sequence of a
protein is of limited biological relevance without some knowledge of both its structure and its
function; protein structures provide a wealth of information that cannot be deduced from their
primary sequence alone; therefore, we can get a complete understanding of protein roles by
analyzing them in structural terms. Structure-based methodologies are consequently regarded as
more robust than sequence-based ones.
The limiting step for these structure-based methodologies is actually having the structure of
a protein at hand. Due to the ever-increasing gap between known protein sequences and structures
and the ever-growing number of protein structure prediction methods available, which are
becoming more and more accurate over time, the use of protein structure models is mandatory.
However, and in spite of progress in the field of protein structure prediction, computed
models often contain structural inaccuracies in both backbone and side-chain spatial coordinates;
instead of being discarded, these models can provide important insights into the function of the
native counterpart; this, in turn, demands the existence of robust methods that can effectively make
use of computed models in the midrange and low range of accuracy, routinely produced by
proteome-scale protein structure modeling projects. Any structure-based algorithm that does not
require high-resolution structures will prove to have a big advantage and an inestimable practical
value.
ModelDB, the tool introduced here, strives to serve as a resource to test any structure-based
method (such as an active site or ligand-binding site predictor) on protein structure models of
different quality. This has the final goal of benchmarking the applicability of protein structure
models for a given novel algorithm. ModelDB builds sets of models of decreasing quality, which
we call decoys, given the sequences experimentally determined proteins. A decoy is a computergenerated protein structure that possesses some characteristics of native proteins, but is not
biologically real. Our system is implemented in such a way that any structure-based existing
method can be tested on the real structure and on the decoy models. The next step is to
automatically assess at which level of quality the results of the tested method differ from those
obtained with the native structure.
Each decoy model is directly compared to its corresponding native structure and precise
quality scores are computed. For a visual insight on how models of different qualities look like and
differ from the native counterpart in a spatial context, they are "colored" following different colorschemes defined by the following spatial descriptors: Solvent accessibilities, secondary structures,
cavity occurrences, average depths, protrusion indexes or burial indexes. This, in turn, allows an
easy visualization and understanding of these parameters' variations in the protein structural
context. Besides, functional annotation is provided when available, in terms of catalytic sites,
ligand-binding sites and other sites of relevance like glycosylation sites.
The tool is publicly available either as an on-line tool or a local application for larger
calculations; it makes use of other in-house tools that also exists independently on-line and for local
use. One of these tools, mappON, colors input structures according to diverse descriptors and
outputs a table with the descriptors of selected residues (and those surrounding them); thus, it serves
to analyze properties of key residues in the protein structural context and visually examine the
results. The other, MAP, has some features intended to deal with the common problem in
bioinformatics of mapping sequence residues onto structures, or structure residues onto another
structure.
Very few other public resources exist for readily retrieving decoy sets of protein structures,
and we indeed have no record of any other automated pipeline for producing such decoys in an easy
and user-friendly fashion. Our tool, apart from allowing to build new decoy sets for a given protein
a scientist is interested in, covers many more different proteins representing a bigger portion of the
protein structural space than any other resource. Furthermore, the on-line version has the advantage
to let the user visually inspect and compare all the models of ranging quality for a given protein in
the same spatial frame. The decoy sets are conceived to test structure-based methods and define to
which extent they can make use of predicted protein structure models. However, the functional
documentation, the model quality estimates and the different color schemes allow many large-scale
analyses to be performed as well.King Abdullah University of Science and Technolog
Studi di citotossicità indotta su cellule umane di melanoma, sensibili e farmacoresistenti, da Clorochina ed amino ossidasi da siero bovino in presenza di poliamine
No full text
Ordering, Timeliness and Reliability for Publish/Subscribe Systems over WAN
No full textIn the last few years, the increasing use of the Internet and geo-political, sociological and financial changes induced by globalization,
are paving the way for a connected world where the information is always available at the right place and the right time.
As such, applications previously deployed for ``closed'' environmets, are now federating into geographically distributed systems connected
through a Wide Area Network (WAN). By this evolution, in the near future no system will be isolated: every system
will be composed by interconnected systems, i.e., it will be a System of Systems (SoS). Example of SoS are the Large-scale
Complex Critical Infrastructure (LCCIs), such as power grids, transport infrastructures (airports and seaports), financial infrastructures,
next generation intelligence platforms, to cite a few. In these systems, multiple sources of information generate a high volume of events
that need to be delivered to all intended destinations by respecting several Quality of Service (QoS) constraints imposed by the
critical nature of LCCIs. As such, particular attention is devoted to the middleware solution used to disseminate information in the SoS.
Due to its inherently scalability provided by space, time and synchronization decoupling properties, the publish/subscribe paradigm
is becoming attractive for the implementation of a middleware service for LCCIs.
However, scalability is not the only requirement exhibited by SoS. Several services need to control a broader set of QoS requirements, such
as timeliness, ordering and reliability. Unfortunately, current middleware solutions do not address QoS constraints required by SoS.
Current publish/subscribe middleware solutions for the WAN environment offer only a best effort event dissemination, with no additional
control on QoS. Just a few implementations try to address some isolated QoS policy, making them not suitable for a SoS scenario.
The contribution of this thesis is to devise a QoS layer that can be posed on top of a generic publish/subscribe middleware that enriches its
service by addressing: (i) ordering, (ii) reliability and (iii) timeliness in event dissemination in SoS over WAN. Specifically, we first
analyze several real case studies, by highlighting their QoS requirements in terms of ordering, reliability and timeliness, and compare
these requirements with both current research prototypes and commercial systems. Then, we fill the gap by proposing novel algorithms to
address those requirements. The proposed protocols can also be combined together in order to provide the QoS level required by the particular
application. In this way, QoS issues do not need to be addressed at application level, so as to leave applications to implement just their
native functionalities.The thesis addresses timeliness, reliability and ordering issues in publish/subscribe systems over WAN. It comes along with a description of the designed protocols and an experimental evaluation of their performance