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Innovative approaches for rapid antemortem diagnosis of prion disorders and to predict synergistic effects of drug combinations
Get PDFThe treatment of cancer is mostly based on a multidrug strategy. The administration of more than one
drug at the same time aims to avoid the increase of drug resistant cells and reduce the dose of each drug
of the combination, exploiting the synergistic effect. At the moment the evaluation of the synergistic
effect is mainly performed using the Combination Index (CI) method and also Isobolograms (IB). The CI method is based on the assumption that the effect of the drugs is due only to the inhibition of enzyme kinetics (Chou et al. 1981). This assumption is not valid for any kind of drug in use; it is well known and studied that also other mechanisms can be involved. Cisplatin (CDDP) is one example of anticancer drug that has a different mechanism of action than direct effect on enzymes. Moreover, both CI and IB
methods can only establish the effectiveness of the drug combination experimentally tested. We tried to
overcome the limits of the classical approach using the Experimental Design (ED) and Artificial Neural
Networks (ANNs); this approach, in fact, could not only allow to better quantify the degree of drug
synergism of the tested combinations, but also to predict the effect of all possible drug doses, leading to
the identification of the combination with the most powerful synergistic effect.
An ANN is a powerful mathematical tool in the form of a computer software that recreates the structure
and the functions of a biological brain. It is composed by logic units called “neurons” organized in 3 layers: input, hidden and output. After an appropriate “learning” process the ANN is able to predict the response for values of inputs never used before. The learning process is performed with a limited number of experiments that produce input data for the network called “training set”.
A previous study (published by Journal of Inorganic Biochemistry) allowed us to evaluate anti-cancer activity
of new copper(II)-phenantroline complexes (Pivetta et al. 2011) versus human hematologic (CCRF-CEM and CCRF-SB) and solid tumor-derived cell lines (K-MES-1 and DU-145); afterwards, ANN method was applied to binary mixtures of these new copper(II) complexes with CDDP tested against CCRFCEM.
The latter study was performed thanks to the joined efforts with a group of chemists of the University of Cagliari and from the Masaryk University (Brno, Czech Republic). The results obtained confirmed our hypothesis and were recently published by Talanta (see following articles)
Precondizionamento ischemico non migliora la prestazione in atleti mezzofondisti in una prova sui 5000 metri di “self paced exercise”
Get PDFThe ischemic preconditioning (IP) is a protective phenomenon against cell injury induced by
infarction. Furthermore, muscle IP has been found capable of improving exercise performance in
laboratory testing. This investigation aimed at verifying the hypothesis that IP improved
performance during self-paced exercise (SPE) in the field. 11 healthy male well trained runners
performed three randomly assigned 5000 m self-paced running test in a regular athletic track. One
was the reference (RT) test, while the others where performed after muscle IP (IPT) and a control
sham test (ST). Average speed was measured during each test. Moreover, mean values of heart rate
(HR), oxygen uptake (VO2), aerobic energy cost (AEC), and blood lactate (BLa) after race were
gathered. We found that none of the studied variables were affected neither by IPT nor by ST with
respect to the RT test. In detail, speed was on average 4.78±0.49, 4.84±0.85, and 4.53±0.38 m·s-1
for the RT, the ST, and the IPT test respectively. HR was 176.4±8.8, 173.8±5.4, and 175.4±5.8
bpm. VO2 was 3.5±0.69, 3.74±0.85, and 3.62±1.19 l·min-1. AEC was 1.04±0.15, 1.08±0.1, and
1.09±0.15 kcal·kg-1·km-1. Finally, BLa after races reached a level of 12.85±3.54, 11.88±4.74, and
12.82±3.6 mmol·l-1. Findings of the present investigation indicate that ischemic preconditioning is
not capable to ameliorate performance during self paced exercise in the field. These results are in
contrast to what previously found in the laboratory setting
Impact of dietary conjugated linoleic acid (CLA) on fatty acid metabolism and endocannabinoid biosynthesis
Get PDFBackground: Conjugated linoleic acid (CLA) refers to a group of positional and
geometric isomers of linoleic acid (LA) mainly found in the meat and dairy products of
ruminants. CLA has been shown to possess different biological activities such as anticarcinogenic
and anti-atherogenic properties, and also to influence body weight, energy
and lipid metabolism, immune response, and inflammation. The endocannabinoid
system (ECS) is involved in a variety of physiological processes, including the
regulation of feeding behavior and energy homeostasis. ECS includes neuromodulatory
archidonate-based lipids, the best characterized are 2-arachidonoyl-glycerol (2-AG) and
N-arachidonoylethanolamine (AEA or anandamide), which activate specific
cannabinoid receptors (CB1 and CB2). N-oleoylethanolamide (OEA) and Npalmitoylethanolamide
(PEA) are generally termed as endocannabinoid-related
compounds. OEA has been shown to decrease food intake and body weight gain, while
PEA is known to possess anti-inflammatory activity. Conversely to endocannabinoids,
OEA and PEA don’t exert their actions by activating cannabinoid receptors, but mostly
through binding the peroxisome proliferator activated receptors (PPARs).
Methodology: The aim of the present thesis was to evaluate the effects of dietary intake
of CLA on fatty acid metabolism, tissue fatty acid incorporation and endocannabinoid
biosynthesis in liver of obese Zucker rats and in plasma of healthy volunteers. 32 male
rats were fed with an equimolar mix of c9,t11 and t10,c12 CLA isomers (about 1%)
supplemented or not with two different background diets, one based on fat of vegetal
origin, PO, and the other made with a fat content of animal origin, OF. Treatment lasted
3 months. In a randomized cross-over study we also evaluated the effects of very high
doses of CLA on lipid profile and endocannabinoid levels in plasma of 24 healthy men
and women (aged 18 to 65) who consumed each three distinct diets for three weeks.
Diets were identical except for 7% of total energy (approximately 20 g/day) which was
provided either by: CLA isomers (80% c9,t11 and 20% t10,c12), industrial trans fatty
acids, or oleic acid (OA).
Principal Findings: In Zucker rats, irrespective of the background diet CLA affected
body and liver weight, with a reduced hepatic lipid deposition. CLA intake increased
concentration of arachidonic acid (AA) with both PO or OF diets, and docosahexaenoic
acid (DHA) only when CLA was included in the OF diet. CLA feeding in combination
with PO diet only, increased retinol level. CLA intake resulted in a decrease of �9
desaturase index, which was inversely correlated to n-3 highly unsaturated fatty acid
(HUFA) score in OF diet. In human plasma, CLA compared to OA diet decreased the
metabolism and the incorporation of the LA metabolites, and influenced alpha linolenic
acid (ALA) metabolism, which resulted in an increased n-3 HUFA score. Also, we
confirmed as high doses of CLA reduced elongation of long chain fatty acids (LCFAs).
CLA intake did not cause significant changes in the profile of endocannabinoids in liver
of Zucker rats and in human plasma, but we found increased levels of PEA in OFCLA
fed rats, and OEA in both OF and PO background diets supplemented with CLA.
Conclusions: Based on our results, we conclude that the effects of CLA on fatty acid
metabolism and endocannabinoid biosynthesis are strongly influenced by the
background diet, which may also explain the differences found between experimental
animals and humans. By improving n-6/n-3 HPUFA balance and sustaining PPAR�
activity, directly and indirectly through OEA and PEA, CLA may exert its beneficial
actions on human health and protection against some diseases, especially those
characterized by chronic inflammation due to an impaired body fat deposition
Il ruolo della vegetazione sui processi idrologici di ecosistemi Mediterranei in condizioni idriche limitanti
Get PDFClimate change, human influences (deforestation, overgrazing, urbanization and pollution) and increased pressures on water supplies by civil and industrial utilizations are affecting the water availability in semi-arid Mediterranean regions. For instance, in Sardinia water resources supply decreased significantly in the last decades, such as shown by annual input from stream discharge into the reservoir system, which are the major source of water for the Sardinia Region. This happens for example in the Flumendosa reservoir system which constitutes the water supply for much of southern Sardinia, including the island's largest city, Cagliari. For this reason, in this study the fundamental processes (e.g., evapotranspiration and surface runoff) of the hydrologic balance have been studied extensively throughout the use of modern techniques for field site monitoring and the support of numerical models. In the first part of this work the evapotranspiration process in a typical Mediterranean ecosystem has been studied through the coupled use of sap flow sensor observations and eddy covariance measurements. Results show the key role of sap flow technique for the estimate of ET in such dry conditions, typical of Mediterranean ecosystems. In the next step the contrasting influences of vegetation differences (grassland versus mixed ecosystems) and soil differences (deeper alluvial valley soils versus shallow upland soils) on evapotranspiration (ET) and CO2 exchange dynamics have been examined. Data from two representative case study sites within the Flumendosa river basin on Sardinia were obtained. At both sites, land-surface and CO2 fluxes were estimated by eddy covariance instruments on micrometeorological towers. Fluxes at the two ecosystems were compared, and the effect of the vegetation cover was examined with the help of an ecohydrologic model to control for the different soil influences. The results show that the water and carbon fluxes in these ecosystems are more controlled by soil differences during the late spring, when the deeper soil depth leads to a doubling of the available moisture and an increase of 48% in the mixed natural vegetation transpiration. The system then switches to vegetation control in the summer as the presence or absence of drought-tolerant trees is the dominant imprint on continued transpiration and photosynthesis. In fact, total grassland ET in the summer is only 20% as large as the mixed vegetation ET in the summer. Finally the study of the surface runoff process have been developed experimentally in the Orroli field site to assess the runoff response of the land surface with varying vegetation states to ultimately predict how changes in the climate of Mediterranean watersheds may affect the needs of water resource management. For this purpose a 4 m by 4 m rainfall simulator was designed, constructed, and tested as the first stage of this research. The rainfall simulator consisted of four independent lines of low-cost pressure washing nozzles operated
at a pressure of 80 mbar, with the number of nozzles determining the rainfall intensity delivered to the plot. The rainfall intensity of the simulator varies from approximately 31 to 62 mm/h with a coefficient of uniformity ranging from 0.62 to 0.75. Measurements taken include surface runoff using a tipping bucket flow meter and soil moisture throughout the plot. The simulator was used to monitor changes in the surface runoff throughout the seasons (July 2010, August 2010, June 2011, July 2011, December 2011, January 2012, May 2012) as the vegetation changes. Results shows the great impact of the changes in vegetation cover on soil runoff process: the vegetation cover is an important factor governing the infiltration process and consequently the surface runoff. The increase of vegetation cover induces large infiltration rates and it drastically reduces the surface runoff amount
Introduzione alla veicolazione transdermica dell’Ibuprofene Sale Sodico e quantificazione in HPLC
Get PDFThe aim of this work is to quantify the Ibuprofen sodium salt in samples derived from Franz cells transdermal delivery experiments.
An analytical method for a determination of Ibuprofen sodium salt and Diclofenac sodium salt (used as internal standard) has been developed, assuring rapidity, accuracy, precision, selectivity and a minimal treatment of the sample.
With this method we have been able to analized not only the sample obtained from the recipient cell, but also the skin and the formulation used in the experiment.
To this aim a methods that would allow the easy removal of the drug from the tissue has been developped.
In this way, it was easy to verify the real quantification of the drug in all phases of the experiment and quantify its total delivery.
In order to perform High Performance Liquid Cromatography (HPLC) analysis, human skin used in experiment has been dissolved first and then deproteinized.
The effectiveness of deproteinization performed through methods such as precipitation with organic solvent, cryo-treatment, Amicon filter tubes, HybridSpe cartridges and Folch method has been verified through the use of the NanoOrange kit.
For the HPLC method, the working conditions were as follows: RP18 column EC 150/4,6 NUCLEODUR® 100-5 C18 ec, Macherey-Nagel GmbH & Co. KG protected with a guard column (EC 4/3 NUCLEODUR®, 100-5 C18 ec, 100-5 18 ec, Macherey-Nagel GmbH & Co. KG) and a pre-column (cut-off: 0,2 μm, Supelco, n. cat. 55215-U); mobile phase phosphate buffer 25 mM - Acetonitrile (73:27 v/v) (pH 7) adjusted with hydrochloric acid; flow 1.0 mL/min.; injection volume of 20 μL; and UV detection was carried out at 265 nm, and experiment were performed in a room where temperature was constantly kept at 25°C.
With this method, the detection of a drug concentration in a range between 0.03 - 7.69 μg/mL has been allowed.
Therefore, the proposed method can be successfully used for drug analysis in samples derived from transdermal delivery experiments
Dust modeling and evolutionary implications
Get PDFFrom the beginning of the 20th entury astronomers started to move their studies toward that parti ular omponent whi h ould be responsible of the reddening and the extin tion of stars radiation. They agreed that this omponent must be represented by a population of mi ros opi parti les named interstellar dust, that it must be mixed with the interstellar gas. Later on, in the se ond half of the entury, dust was re ognized to play very many important roles in the Milky Way and other galaxies. These ndings hanged the astronomer's view of the interstellar spa e, that is now fully onsidered as an a tive element in gala ti evolution. For this reason to a hieve a more reliable understanding of the nature of interstellar dust is ne essary to take into a ount the response of dust to the physi al onditions of the regions in whi h dust grains are embedded. Over the years astronomers used the variation of extin tion respe t to wavelength, the interstellar extin tion urve, to study osmi dust. In this thesis I elaborate a physi al and hemi al model of dust, to investigate the remarkable variety of gala ti interstellar extin tion urves. In Chapter 1 I introdu e the roles of the interstellar dust, its pro essing in the interstellar medium, and the tools to address the problem of its nature. In Chapter 2 I present the exploited model in a simpli ed version, and an appli ation to some pe uliar lines of sight in our galaxy. In Chapter 3 the omplete version of the model is put forward, and, together with its simpli ed version, applied to a large sample of Milky Way interstellar extin tion urves. All di erent avours of observed extin tion urves, ranging from the average gala ti extin tion urve to very pe uliar pro les, an be des ribed by su h a model. I show that a mixture of ore mantle sili ate/ arbon grains together with a relatively small number (54 spe ies in four harge states) of poly y li aromati hydro arbons an reprodu e the features of the extin tion urve in the ultraviolet, dismissing an old obje tion to the ontribution of poly y li aromati hydro arbons to the interstellar extin tion urve. In Chapter 4 I ompare an evolutionary model of the physi al properties of arbona eous grain mantles with their determination through the tting pro edure outlined in Chapter 3. The results of su h omparison demonstrates, that in the framework of the adopted dust model, the whole sample of gala ti extin tion urves are in striking agreement with the proposed evolutionary s enario, requiring physi al onditions fully onsistent with standard values of density, temperature, radiation eld intensity, and average age of di use interstellar louds. The results of this thesis show that the present model is able to re on ile the great variety of observed interstellar extin tion urves within an uni ed evolutionary s enario, without making drasti hanges to stru tural properties of dust grains
Characterization of Human Endogenous Retrovirus sequences identifed in the human genome using the RetroTector software
Get PDFHuman Endogenous Retroviruses (HERVs) represent the inheritance of ancient germ-line cell infections by exogenous retroviruses and the subsequent transmission of the proviral integrated elements to the descendants. Actually, no replication-competent HERV sequence is recognizable in the human genome. However, some HERVs retain one or several intact retroviral genes and may express protein products that could interfere with the human immune system. The number and classification of
HERVs vary according to method of enumeration. The focus of this project is to perform a systematic analysis and a classification of the most intact HERV sequences in order to better understand their evolution and their involvement in shaping the human genome.
The human genome assembly GRCh 37/hg19 was analyzed with
RetroTector software and a total of 3290 HERV proviral sequences were identified.
The complex genetic structure of the 3290 proviruses was resolved through a multi-step classification procedure that involved a novel type of similarity image analysis (Simage). The 3290 HERV were classified in 40
unique clades (groups) which could be placed into class I (Gamma- and Epsilon-like), II (Beta-like) and III (Spuma-like). Simage analysis contributed to define the presence of a high number (around 40%) of mosaic forms, with heterogenous sequence content.
A finest characterization of the HERV sequences was achieved with the investigation of a broad panel of structural markers that contributed to confirm and extend the previously performed classification.
Finally, the HERV background of integration was also studied. Integration patterns analysis showed a tendency for proviruses from the same clade to occur together, within 100000 bases, maybe due to local duplications.
Representatives from some gammaretroviral clades (HERVH and HERVE) integrated more frequently than expected by chance into the 5´end of transcriptional units, mostly in antisense orientation. A few lncRNAs were also found to contain HERV sequences. Thus, cis-effects from HERVs are to
be expected.
In conclusion, this study represents an advance in the state-of-the art of HERV characterization within the human genome and a starting point for upcoming studies on HERVs
Possibile ruolo del sistema endocannabinoide nel disturbo d’alimentazione incontrollata (binge eating disorder): studi comportamentali, farmacologici e biochimici
Get PDFAccording to the Diagnostic and Statistical Manual of Mental Disorders (DSM-V), binge eating disorder (BED) is an eating disorder characterized by repetitive episodes of uncontrolled and excessive food consumption (binge eating), in a short period of time, without the inappropriate
compensatory behaviors for limiting weight gain. BED is a stable condition that is associated with
elevated psychiatric comorbidity, including depression and anxiety.
A large body of evidence supports a contribution of the endocannabinoid system in the modulation
of energy balance by controlling food intake through central and peripheral mechanisms. The cannabinoid CB1 receptor is abundantly expressed in the central nervous system and induces inhibition of neurotransmission through modulation of presynaptic neurotransmitter release. CB1
receptors inverse agonists inhibit food intake through both central and peripheral mechanisms while, in contrast, cannabinoid agonists stimulate food intake in humans and induce beneficial effects in acquired immune deficiency syndrome related to anorexia, suggesting altered
endocannabinoid neurotransmission in anorectic conditions. A possible involvement of the endocannabinoid system in the pathogenesis of BED and other eating disorders has recently been supported by independent experimental evidence. Elevated plasma levels of anandamide were found in women affected by AN and BED. Moreover, anandamide levels were inversely correlated with plasma leptin concentrations.
On the basis of these evidence, the aim of this thesis was to study whether and which elements of the endocannabinoid system might be correlated with the binge eating behavior, which brain areas are specifically involved, and if pharmacological treatments specific for the endocannabinoid
system (i.e. agonists, antagonists, inverse agonists of cannabinoid receptors, inhibitors of endocannabinoid metabolisms) are be able, besides to modify the state of BED induced in laboratory animals, to restore a correct functionality of the endocannabinoid system.
Furthermore an analysis of the behavioral profile of animals with BED has been evaluated before
the start of drug treatment by means of different mazes in which is possible to study anxiety and depression.
Binge eating behavior was induced in animals by giving them a sporadic (3 days/week) and limited (2h) access to a high fat diet (margarine) in addition to a continuous access to chow and water. In these animals, the intake of margarine becomes significantly greater than in animals with limited
daily access to margarine and remains stable over prolonged periods of time. As revealed by forced swim test, animals with binge eating behavior did not show a depressive like behavior compared to control animals. However, using the elevated plus maze, an anxiety like behavior was highlighted in those animals before access to margarine (Pre - binge phase) that it was significantly reduced after
the consumption of this palatable food (Post - binge phase). Results showed that an increase of the
endocannabinoid signaling by CB1 agonists or by inhibitors of endocannabinoid metabolism did not modify the binge eating behavior presents in our animals. On the contrary, a decrease of the endocannabinoid signaling by CB1 receptors inverse agonist/antagonist rimonabant, was able to
alter this behavior when given chronically. As regards the CB1 receptor density, no difference has been highlighted between animals showing binge eating behavior and control group. In conclusion, negative modulation of the endocannabinoid signal may represent an important strategy in the treatment of Binge Eating Disorder
A methodology for evaluating the cost-effectiveness of residential building stocks retrofits in Italy and Denmark
Get PDFBuildings are at the centre of our social and economic activity. Not only do we spend most of our lives
in buildings, we also spend most of our money on buildings. The built environment is not only the
largest industrial sector in economic terms, it is also the largest in terms of resource flow1.
The rising energy costs, the growing concern about environmental issues and the approaching
exhaustion of world energy resources are urging the entire European Community and the several
national governments to improve energy management. Special attention is usually paid to public
administrations, as European and national legislations often point out that these bodies must provide
energy efficiency measures, as well as for the reasons mentioned above, also in order to represent an
example for the entire community and for citizens as well.
But it is also very important to find out how to foster and encourage energy efficiency improvements
and saving measures in private dwellings to achieve the double advantage of reducing the global
energy consumption level within the private sector and increasing investments, favoring the creation
of additional cash flows as well.
The possible combination of such multiple benefits makes the building sector a crucial field for policy
makers at EU and national levels. Hence a policy framework that supports national markets in
unlocking these potentials is strongly needed. With overall European policy aimed at significantly
decarbonizing its economy by 80% to 95% by 2050, the building sector must undoubtedly play a key
role. And any strategy to tackle the challenge in this field will clearly require both a significant amount
of financial investments and long-term political commitments.
The main goal of the present research is to propose an optimized methodology and cost effective
decision-making process - based on the main facts emerging from the adoption of the key energy
policies and financial instruments currently in force at European level (particularly in Italy and
Denmark) - also to outline the next policy steps in improving the energy performance of buildings.
After a global overview of the policies adopted at European level, the analysis focuses on the two
different regulations implemented at the national level by Italy and Denmark. Furthermore, to define
the best mixture of energy retrofit measures for the different geographical areas of Italy - applying a
methodology based on simple and available data to improve residential buildings' energy efficiency -
the work started with the analysis of the several reports produced by ENEA (the Italian Research
Agency for Energy Efficiency) since year 2007. These were based on the data collection performed in
order to assess the effectiveness of the Italian government’s financial policies established to support
energy saving actions in private dwellings.
The first steps of such a top-down analysis are then carried out both through manual cost/benefit
spreadsheets, as well as with the implementation of a linear programming analysis tool.
The study defines different linear programming models, depicting different optimization problems
(e.g. energy saving maximization vs. retrofit cost minimization), along with the respective different
background scenarios. Such investigations are therefore carried out through the implementation and development of Dantzig's
simplex algorithm.
Moreover, to carry out a global comparison between the overall Italian and Danish situations, also
achieving a deeper single-dwelling-focused analysis, further studies are developed through a Building
Energy Optimization tool, implementing the EnergyPlus dynamic energy simulation software.
Thence, the research moves on to a more specific analysis, shifting to a bottom-up approach and
involving in the enquiry a comparison between the different assessment settings (climatic, political,
economic, cultural) depicted both by Italy and Denmark.
Two different dwelling models are defined for the above countries, focusing the analysis on those
building typologies most representative of such European nations and thence different retrofit
solutions are depicted and analyzed.
The results obtained by means of this dynamic assessment are then used to group the respective energy
savings vs. retrofit cost considerations within a global cost-effectiveness assessment.
Finally, some “guidelines” are outlined to address the challenge of renovating the existing building
stock, also in order to keep pace with the aims of both the nations and the European Union
L’economia della natura e il respiro del mondo. La filosofia della natura di Swedenborg tra Herder e Schelling (1770-1810)
Get PDFThe central role of the notion of ‘life’ mainly characterises the dialogue between Naturphilosophie and the sciences between the end of the Eighteenth and the first decades of the Nineteenth Century in Germany. Many problems and topics, from the relation Infinite-finite to that between inorganic matter and living beings, rely on this concept. Especially in the period between 1770 and 1810, authors such as Herder, Goethe and Schelling tried to provide a philosophical explanation of the genesis of the cosmos and that of the single organisms. In so doing, they pursued an understanding of how the coexistence of unity and variety in every part of the universe, and the concurrence of the ‘economy’ of the whole and the individual development, are possible. This led them to a dynamic idea of nature: a continual transformation or metamorphosis of forms takes place according to the repetition, in different ways, of a unique model or ‘typus’ and suggests the view of the world as an organism, according to the old formula that ‘the great’ corresponds to the ‘small’. For a better understanding of these problems, it is useful to consider them also from a historical point of view, taking into account some of the sources of the German thought of these decades about life and nature. In this perspective, Emanuel Swedenborgʼs philosophy of nature can be considered as a paradigm for many concepts, all related to the problem of life, which are very important for the German philosophers such as Herder and Schelling. For this reason, I will consider the reception of his ideas and works in Germany during the years from 1760 to 1810, taking advantage, in particular, of the reviews of his scientific and philosophical writings, and of the survey of the presence of his works and translations in the catalogues of the private libraries of philosophers. A big part of this thesis is devoted to the analysis of Swedenborg’s, Herder’s and Schelling’s writings (some of which are not available in Italian language yet), in order to make a comparison and show the connections among their ideas, which still partially need a thorough consideration