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La patologia del provvedimento amministrativo: analisi e sviluppo dell’eccesso di potere
Get PDFNowadays, misuse of power is the most important flaw of administative measures. Its history allows to understand the reason.
Born by French jurisprudence as a non-defined institution, it would be inplanted in Italian jurisditional system by administrative judgements. The interest in misuse of power let it enter in Italian law system.
Jurisprudence, anyway, contribuited to design a variety of form of it.
Also, today misuse of power is enhancing its importance, especially for its appearence in communication and information technologies’contents.
Its development, in recent times, need to be focused. This research try to explain how and why misuse of power is changing, with analysis of relevant case law too
Linking humanitarian and development action in Ethiopia
Get PDFThe purpose of this doctoral research has been to analyse the integration of
humanitarian and development action during the 2011-12 food crisis in Ethiopia.
Historically, the two types of action have evolved as two different, yet intertwined,
domains, with separate objectives and guiding principles. Yet, particularly since the
1990s, there have been debates on how to better integrate them, in consideration of the
fact that they often operate in the same context, as was the case in the Ethiopian casestudy.
Following a neo-institutionalist approach, this study has analysed what hinders
the bridging of the humanitarian-development divide, and what supports it, by
analysing the interplay of institutional forces in the regulative, normative, and culturalcognitive
domains in the Ethiopian case.
Grounded in critical realism and in neo-institutional theory, this research has adopted
a novel theoretical approach to the issue of linking humanitarian and development
action, and has been based on original empirical material. A key finding of this study is
that, in the context of Ethiopia, integrating the two modes of action is possible and
even encouraged at some levels, despite a broader funding architecture that hinders
such linkages. By shedding light on practices of humanitarian relief and development
in Ethiopia, this study contributes to scholarship in the broader fields of International
Development and African Studies
La comunità macrozoobentonica come strumento per la valutazione dello stato di qualità ecologico in ambienti di transizione: il caso della laguna di Santa Gilla
Get PDFTransitional habitats are vulnerable ecosystems, affected by strong and unpredictable environmental parameters and exposed to several threats, both natural and anthropic, that can deeply interfere with the biological components critical to their functionality (Lardicci et al., 1993; Fraschetti, 1996; Giangrande & Mistri et al., 2000; Lardicci et al., 2001,).
The conservation and management of these habitats require an integrated study of three aspects: chemical, physical and biological (Gibson et al., 2000).
Recent directives, specifically Water Framework Directive 60/2000/EC and Marine Strategy Framework Directive 56/2008/EC, suggest that biological elements are essential tools to evaluate the quality of coastal and transitional ecosystems. In particular, the macrozoobenthic component is often employed in transitional environmental programs (Blanchet et al., 2008).
The location analyzed in this study is one of the most important wetlands in Sardinia. Despite this, there is no complete and recent information about the benthic community.
The main aim of this study is to characterize the Santa Gilla lagoon, studying some abiotic aspects (granulometry, mineralogy, heavy metals and organic matter in sediments, and salinity, pH, dissolved oxygen and temperature of the water column) and providing information about the macrozoobenthic community in relation to spatial-temporal variations, creating the research foundation for the development of an appropriate resource management and conservation strategy.
A total of 16910 organisms were found, belonging to 114 different taxa.
The distribution of these organisms in relation to the abiotic parameters was studied. Multivariate analysis highlighted two main groups, originating from distinct areas of the lagoon and influenced by different factors.
However, it is difficult to determine which variable most strongly influences the distribution of the community. Indeed, benthic organisms are expected to respond to a more complex set of environmental factors related to the water column, sediment, interstitial water, and interface layer (e.g. Maurer et al., 1985; Whiteman et al., 1996).
Different biotic indices were applied and their effectiveness was evaluated. The results were varied, but all measurement indices concluded that best ecological quality measured was in close proximity to the sea.
Through this project, important information necessary for future integrated ecosystem management was gathered. Undoubtedly, the analysis of the macrozoobenthic community is a prerequisite for the evaluation and management of ecosystems in a sustainable and appropriate manner (Ludovisi et al., 2013).
Thus, this study constitutes the starting point for future quality status assessments, as well as the basis for subsequent analysis and monitoring
Quality regulation and energy saving through control and monitoring techniques for industrial multicomponent distillation columns
Get PDFThis Thesis deals with the problem of the quality control and monitoring
and the related energy consuming aspects in industrial multicomponent
distillation columns. These have important relevance especially in case of
units subjected to important changes in operating conditions which
manifests in fluctuation of the product concentration.
Due to the low reliability of the seldom available and delayed analytic
composition measurements, they cannot be employed for an efficient
online monitoring of the separation. Instead, this Thesis proposes the use
of composition observers with passive innovation mechanism and driven
by temperature measurements. The passive structure allows to limit the
number of ODEs to be online integrated, which is a central issue to obtain
a product suitable for the industrial implementation. The design of this
passive structure involves the choice of number and location of the
temperature sensors, the choice of the innovated component and the set of
modeled components in the (possibly simplified) estimation model. This
issue is addressed with a simple and systematic methodology that employs
steady state information about the per-component temperature gradient of
the column and detectability measures and conditions. Differently from
the majority of the works in literature, the proposed methodology allows
also to assess the feasibility of obtaining good estimation performance
using the available temperature sensors in real columns.
Even the analytic composition measurements cannot be employed for
quality control in feedback control loops mainly due to delay problems.
Instead, temperature feedback controllers are usually used. But even if the
temperature sensor is well located (and in this Thesis a criterion for sensor
location is suggested based on an extension of the slope criterion to the
multicomponent case), when the operating conditions undergo changes,
the indirect regulation of the product quality cannot be achieved, with
alternating periods of under and over-purification that also adversely
affect the energy requirement for the column functioning. Thus, in this Thesis an application oriented solution is proposed consisting in adding a
feedforward temperature setpoint compensation that guarantee a close
quality regulation, including a more efficient management of the energy,
under the changing operating modes of the column.
The methodologies and techniques proposed and described in this Thesis
are tested with an industrial multicomponent IC4-NC4 splitter located at
the Sarlux Refinery (Sarroch, Italy), which, motivated by the good results,
has already implemented the composition estimator and is going to
implement the feedforward setpoint compensator
Life cycle assessment e water footprint assessment nella progettazione di una filiera bosco-energia in Sardegna
Get PDFThe study of the potential environmental impacts of a hypothetical wood-energy supply chain has integrated: the application of models (particularly, spatial models), the chemical and physical characteristics of the forest biomass, the techno-economic viability of different plant configurations and the Life Cycle Assessment approach, for the sustainable design of a supply chain in its life cycle. The main results are relevant in the decision-making process, related to the installation of the chain. They allow the decision maker to select the most appropriate choices, between multiple scenarios, as well as between various improvement options and responses for the potential impacts reduction. The present study has developed an approach for the wood-energy supply chain design, which integrates its most significant components (the cultural heritage of the territory, the best forestry practices, the technological and economic issues related to the exploitation of forest resources), by interpreting them from the environmental viewpoint. Indeed, for a really sustainable supply chain, none of the abovementioned components should be excluded, being them connected each other. LCA provides a valid interpretation of the systems and product supply chains related to the forest biomass, both in terms of impacts on various environmental matrices, and from that of the related potential damage on the Areas of Protection (human health, ecosystem quality, resources). The methodology is, actually, widely used in ex-post evaluations of wood-energy sectors, but, on the contrary, it is poorly implemented for the feasibility study and design; thus, the present work could represent a reference point and a baseline for future studies, insights and improvements. The study area is the Monte Olia public forest, located in the Northern side of Sardinia Island (Italy) and managed by Ente Foreste della Sardegna. A Life Cycle Assessment has been performed on the potential supply chain, by considering its main phases. Firstly, the availability of forest biomass for energy purposes has been estimated. Then, each phase of the chain has been analysed, by applying average data from similar case studies, as well as by implementing spatial models and optimisation methods for the biomass transportation. The most economically viable plant configuration has been selected by carrying out a techno-economic and environmental analysis on different plant technologies for the energy conversion of woody biomass. It has taken into account the energy demand of the service buildings within the study area, along with the characterisation of the main forest species of the Monte Olia forest (by means of laboratory determinations). The water consumptions and the emissions into water in the Life Cycle Inventory have been used for the second part of the study, which has focused on the application of the Water Footprint Assessment methodology for the designed supply chain. The analyses have consisted in: the quantification and separation of the local water appropriation and of the external amounts; the identification of the most significant supply chain phases with reference to the freshwater appropriation; the comparison between the internal appropriation volumes (related to the annual energy production) and the annual rainfall on the study area. The Water Footprint Assessment results have led us to assert the sustainability of the supply chain, from the point of view of the local freshwater appropriation. Another objective has consisted in the comparison of the Water Footprints related to the specific wood-energy supply chain and to a similar chain fed with fossil fuels (oil-fired boiler, instead of the biomass boiler), by identifying the differences in terms of Water Footprint components. The
results have confirmed the greater environmental sustainability of short supply chains fed with forest biomass, than those fossil fuel-dependent. The last objective has concerned the difference between the impacts on water obtained by LCA and the results of the Water Footprint Assessment. In the present study, Life Cycle Assessment and Water Footprint Assessment have been effective tools for the sustainable wood-energy supply chain design. The use of both methodologies has allowed us to identify some critical points in the use of LCA for the management of the freshwater resources, if not being used in synergy with the Water Footprint Assessment
Bioinformatics e Biostatistics applied to research in pediatric genetic disease. Clinical evidence in IFNλ4 polymorphisms associated with HCV infection in patients with beta thalassemia and WGCNA analysis weighted for IFNλ4 genotype rs12979860 to detect RPL9P18 as hub in HCV infected cell.
Get PDFGenome-wide association studies have identified host genetic variation to be critical
for spontaneous clearance and treatment response in patients infected with hepatitis C
virus (HCV). We demonstrated the same in patients with thalassemia major infected
by genotype 1b of HCV.
In the present first part study we retrospectively analyzed 368 anti-HCV positive
patients with beta-thalassemia in two Italian major thalassemic centers (Cagliari and
Turin).
The strongest IFNλ4 SNP found associated with HCV was rs12979860 where C/C
genotype was related to response to the interferon treatment and, above all, to
spontaneous clearance of the virus. However, the positive predictive power was
stronger for viral persistence than spontaneous clearance indeed TT allele was more
predictive than CC.
Another polymorphism rs4803221 was analyzed because had independent effects
respect to rs12979860. The haplotype tagged by SNP rs12979860 and rs4803221
significantly could improve the viral clearance prediction in infected patients.
Neither necrotic-inflammation or bilirubin values in the chronic phase of the hepatitis
C were related to IFNλ4 polymorphisms. No relation among IFNλ4 polymorphisms
and fibrosis stage directly shown by the liver biopsy was found.
Second part of our study was to identify hub genes associated in pathways closely
related to IFNλ4 variants in HCV response. We used gene expression profile data of
GSE54648, downloaded from Gene Expression Omnibus (GEO). We focused our
attention on expression genes differential between rs12979860 unfavorable TT
genotype and favorable CC genotype, using weighted gene expression network
analysis (WGCNA - R package). Significant modules were selected using the
clustering analysis. At the final the best significant module was “black” module. Its
pathways were involved in translation mechanisms such as translation termination,
translation elongation, nuclear-transcribed mRNA catabolic process, cellular protein
complex disassembly, therefore biological mechanisms that occur inside ribosome.
We discovered RPL9P18 pseudogene as a hub potentially related in inhibition of
spontaneous clearance and furthermore likely involved in drug treatment inhibition.
Our result suggests an active role for ribosome pseudogene in innate antiviral
response probably during ISG (IFN-stimulated genes) translation. Moreover, through
co-expression analysis we demonstrate a new possible role of IFNλ4 genotype in
HCV infection, associate with expression of ribosomal pathways
Progettazione e sintesi di nuovi antagonisti CB1 a struttura pirazolica come potenziali radiofarmaci per la PET imaging
Get PDFCannabinoid receptors are members of the large family of G-protein
coupled receptors. Two types of cannabinoid receptor have been discovered: CB1
and CB2. CB1 receptors are localised predominantly in the brain whereas CB2
receptors are more abundant in peripheral nervous system cells. CB1 receptors
have been related with a number of disorders, including depression, anxiety,
stress, schizophrenia, chronic pain and obesity. For this reason, several
cannabinoid ligands were developed as drug candidates. Among these ligands, a
prominent position is occupied by SR141716 (Rimonabant), which is a pyrazole
derivative with inverse agonist activity discovered by Sanofi-Synthelabo in 1994.
This compound was marketed in Europe as an anti-obesity drug, but subsequently
withdrawn due to its side-effects. Since the relationship between the CB1
receptors’ functional modification, density and distribution, and the beginning of a
pathological state is still not well understood, the development of radio-ligands
suitable for in vivo PET (Positron Emission Tomography) functional imaging of
CB1 receptors remains an important area of research in medicine and drug
development. To date, a few radiotracers have been synthesised and tested in
vivo, but most of them afforded unsatisfactory brain imaging results. A handful of
radiolabelled CB1 PET ligands have also been submitted to clinical trials in
humans. In this PhD Thesis the design, synthesis and characterization of three
new classes of potential high-affinity CB1 ligands as candidate PET tracers is described
Analisi comportamentale della scelta del percorso attraverso l'utilizzo di nuove tecnologie di acquisizione delle informazioni
Get PDFIn travel demand modeling, route choice is one of the most complex decision-making
contexts to understand and mathematically represent for several reasons. Firstly, a large
number of available paths may exist between the same origin-destination (OD) pair.
Secondly, neither the traveler nor the modeler are aware of all the available alternatives.
Thirdly, individual choices are dictated by different constraints and preferences that are
difficult to capture by modelers who face increasingly larger datasets where retrieving the
exact path chosen by travelers is not always straightforward. Last, there is a lot of
uncertainty about travelers’ perceptions of route characteristics as well as other
characteristics that can influence their choices, such as age, gender, habit, weather
conditions and network conditions. This highlights the difficulties encountered for
interpreting individual user behavior in greater depth. The rapid advances in GPS devices,
has resulted in major benefits for data collection, which now can be recorded automatically
and with greater accuracy compared to the techniques used in the past (phone calls, e-mails,
face-to-face interviews, laboratory experiments.).
On these basis the main objective of the thesis is then to study route choice using a GPS
database. The data were acquired during a survey, named "Casteddu Mobility Styles” (CMS),
conducted by the University of Cagliari (Italy) in the metropolitan area of Cagliari between
February 2011 and June 2012. Each participant was asked to carry a smartphone with builtin
GPS in which an application called “Activity Locator” – implemented by CRiMM (Centre for
Research on Mobility and Modeling) – had been installed. A total of 8831 trips were recorded
by 109 individuals, of which 4791 referring to the car driver mode. Each GPS track
(consisting of a sequence of referenced position points) was then treated with map-matching
techniques, through which it was possible to associate each “GPS point” to a link of the
network, thus creating the observed route database.
The first objective of the thesis is to understand which are the characteristics of the data
acquired during the CMS survey, doing firstly the same analysis that other authors did in
their researches based on GPS data. In almost all the previous researches, the GPS data were
collected through in-vehicle surveys that make it possible to gather objective information on
trips (travel times and distances). Pre-and post-analysis interviews were conducted to
gather information about the subjective characteristics of the individuals and GIS platforms
were used to study the routes. In the present study, the data were collected using an
integrated system able to also record the activities conducted, along with all the
characteristics associated thereto. In this way a complete database was created containing all
the information (objective and not) concerning the trips. For comparisons with the
objectively most convenient paths, then, was used a static macrosimulation model
(implemented in CUBE, Citilabs Ltd.) of the entire study area (Cagliari and its metropolitan area), which reproduces the network characteristics actually encountered by the drivers
referring to the data used.
From this first analysis it was observed that when more than one route is taken for repetitive
trips between the same OD. In order to understand these particular behavior of users, named
also intravariability, discrete choice models were estimated. It’s important to note that in the
previous GPS-based researches this particular behavior was only identified, without
studying it in depth. Several other studies, focused on route switching behavior, tried to
understand it applying discrete choice models, but their database were based on data
acquired through questionnaires or laboratory experiments, and for the majority the route
switching behavior was studied in relation to the trip information provision. The objective of
this analysis is then to combine the two fields of the research on route switching, trying to
understand it estimating discrete choice models using a GPS based database, closing the gap
of the previous researches. The final goal of the model estimations is to understand which
are the main attributes of the routes and the characteristics of the users that most influence
the choice of an habitual route for the same origin-destination (OD) trip.
After these first analysis, the final objective of the thesis is to apply a route choice model to
GPS-based data. Modeling route choice behavior is generally framed as a two-stage process:
generation of the alternative routes and modeling of the choice from the generated choice
set. The focus of this step of the research is on the bias that might be introduced in the model
estimation by the choice set generation process. Specifically, although several explicit choice
set generation techniques are found in the literature, the focus is on stochastic route
generation and the correction for unequal sampling probability of routes when applying this
technique that is easily applicable to large-scale networks. Indeed, stochastic route
generation is a case of importance sampling where the selection of the path depends on its
own properties, so route choice models based on stochastic route generation must include a
sampling correction coefficient that accounts for the different selection probability. In this
study is proposed a methodology for calculating and considering this correction factor into
MNL-based models with choice sets generated by means of stochastic route generation.
Specifically, was decided to look at the sampling correction factor proposed for the random
walk algorithm and to calculate the route selection probability in order to exploit this
expression. Therefore, a procedure is proposed for the computation of the selection
probabilities on the basis of the stochastic generation principle, then the correction factor
and last the EPS for model estimation. The modeling analysis confirms the functionality of
the proposed approach that has great advantages: (i) it provides insight into the application
of stochastic generation in route choice modeling, especially in large-scale networks where
the only need is a standard random number generator and a Dijkstra algorithm; it
proposes a simple and manageable procedure from the computational perspective for the calculation of route selection probabilities and hence the correction factor and EPS for model
estimation; it proves the efficiency of the proposed methodology on revealed preference
data in a dense urban network by showing an increase in goodness-of-fit of the model and a
shift from illogical to logical sign in parameters estimated for key variables such as travel time
La valutazione della risposta cardiometabolica durante l’attività di equitazione
Get PDFHorse riding changes its meaning over time.
Formerly horses were considered just for transports of humans and things.
In last centuries they became hobbies and team mates for equestrian activities.
Horse riding today includes several disciplines, and many of these are olimpic sports.
As such, equestrian deserve the attention of sports researchers, who are interested in cardiometabolic adaptations during training and competition.
Cardio-Pulmonary Exercise Test
The cardio-pulmonary exercise testing (CPET) today is the most reliable test that provides data on cardio metabolic and ventilatory function during exercise.
Data derive from the analisys of :
VO2: oxygen uptake (ml/min);
VCO2: carbon dioxyde production (ml/min);
VE: pulmonary ventilation (L/min);
HR: heart rate (bpm);
It became fundamental to obtain and understanding of physiological capacities underlying sports performance.
It would be useful to know the energy demands and whether the anaerobic metabolism is recruited.
CO2 excess
To obtain an index of anaerobic glicolysis, excess of carbon dioxyde production (CO2 excess) was assessed as follow:
CO2 excess = VCO2- (RER rest ? VO2)
WALK TROT CANTERING
It is quite well correlated with the rate of lactate accumulation in the blood . (Hirakoba et al. 2006, Yano et al. 2002)
It allows to continuous measuring of the recruitment of lactacid metabolism during exercise, without stopping. (Crisafulli et al. 2009)
VO2, VCO2, VE e HR were recorded during a training session in which riders performed four gaits: walk, trot sitting, trot rising and canter. Data collected were subsequently compared to same parameters gathered during a previous incremental cardiopulmonary test on cycloergometer.
However few studies described the cardiometabolic response during riding:
Westerling’s study
Westerling’s study
VO2 varies according to different equine gaits, ranging between 40% and 80% of rider?s maximal aerobic power. The aerobic demands during riding test were high and corresponded to the oxygen uptake at a workload of 120 Watt in the bycicle ergometer test.
Devienne and Guezennec's study
Rider’s mean energy expenditure when riding was between 25 and 70% of VO2max.
Different gaits require more energy expenditure by the rider because they need to work harder to maintain the centre of mass.
Our study
Ten riders were enrolled. They underwent two different tests:
A) Cardiopulmonary exercise test (CPET): a standard incremental bicycle ergometer test until exhaustion on an electromagnetically braked cycle ergometer. VO2, VCO2, VE, and HR were measured by a gas analyzer (Ultima CPX, MedGraphics, USA).
B) Riding session (RS): after 3 minutes of rest to collect baseline data, riders performed 5 minutes of walking, 10 minutes of trot, and 5 minutes of cantering. Throughout RS, VO2, VCO2, VE and HR were collected by a portable telemetric metabolimeter (VO2000, MedGraphics, USA).
VO2 and VCO2 mean values gathered during RS were below the maximum and anaerobic threshold level reached during CPET.
Futhermore all gaits required an energy expenditure significantly higher respect rest and the previous gait.
MATERIALS AND METHODS
parties Involved
Nineteen athletes-riders between the ages of seventeen and forty
years (mainly practitioners sporting-competitive), after giving
informed consent, were involved in the testing and
They are subjected to two different tests: one in the laboratory and the other on the field of
horseback riding.
EXPERIMENTAL PROTOCOL
Cardiopulmonary exercise testing
Exercise testing cardio-pulmonary (CPET) today it is the most reliable test to
to provide data cardiometabolic and lung function during
exercise.
Data derived from the analysis:
• VO2: oxygen consumption (ml / min);
• VCO2: carbon dioxide production (ml / min);
• VE: pulmonary ventilation (L / min);
• HR: heart rate (bpm);
This test has become essential for optimum sports performance
and for the understanding of the physiological capabilities of the base.
From the values of respiratory gases, measured during the CPET, you also get a
useful capacity index anaerobic lactic-athlete.
To obtain an index of use of anaerobic metabolism is measured
the excess production of carbon dioxide (CO2 excess) via the
following equation:
Excess CO2 = VCO2- (RER rest • VO2), where RER stands for respiratory
ratio calculated as the ratio VCO2 / CO2:
!
19!
!
[CO2 = excess VCO2- (RER rest • VO2)]
The graph of the figure below shows the trend of the excess CO2 in
according to the different conditions of motion of the horse nell'equitazione (data
laboratory).
Previous experiments have shown that the excess CO2 correlates well with
the rate of accumulation of lactate in the blood (10, 11). Moreover, this parameter
allows continuous measurement of recruitment of lactate metabolism
during exercise, without the need to stop in order to perform the
blood samples necessary for this assessment (12).
Instrumentation
Metabolimeter Laboratory (CPX, Medgraphics, USA) to measure
breath for breath: VO2, VCO2, Ve.
Metabolimeter portable and wearable (VO2000, CPX, Medgraphics, USA)
for the measurement of breath-by-breath: VO2, VCO2, Ve and frequency
Heart rate (HR).
Experimental session conducted in the laboratory
Each athlete recruited performed a cardiopulmonary exercise testing to
ergometer (CS), with incremental load until exhaustion (13).
During the test they were detected values breath for breath:
VO2,
VCO2,
VE,
HR,
through the use of metabolimeter CPX, Medgraphics, USA.
Experimental session conducted on the field
After a week of laboratory tests, each athlete recruited
He performed a test drive of the horse (RS) with the following protocol:
1) Implementation of the control test consisting of 3 minutes of rest, for
collecting basic data,
2) Perform a 5 minute walk,
3) Perform a 10-minute jog,
4) Make a 5-minute gallop.
During the test RS were acquired the values of:
VO2,
VCO2,
VE,
HR,
through metabolimeter wearable VO2000 - Medgraphics 2000 li
He sent via radio to the stationary unit (14).
Data processing
For each trial phase, in the two sessions of tests performed,
It was calculated the mean value ± the standard error (SE) of each
variable considered in each subject tested.
The average values were then compared with each other via the statistical test
Analysis of variance (ANOVA) and differences were considered
statistically significant for P <0.05.
EXPERIMENTAL RESULTS
In figure N ° 1 the vertical columns represent the average values ± ES of
oxygen consumption (VO2 on the ordinate axis) during the various stages in
which consisted of riding the test in the field, while the horizontal line
dotted in red it represents the average value of maximum consumption
oxygen (VO2-max) achieved by athletes during exercise testing
incremental cycle ergometer, implemented in the laboratory. The horizontal line
dotted in black it represents the value of the oxygen consumption
corresponding to the anaerobic threshold (VO2-AT) during the experiment incremental.Conclusion
Present findings demonstrate that each of the different gaits of equine movement requires different metabolic response
It appears that, while walking and trotting involve predominantly the aerobic metabolism, anaerobic glycolisis is also recruited during cantering
This fact probably reflects the diverse pattern of riders' muscle contraction during each of the specific equine movement
Computational Study on MFP MexA in MexAB-OprM and RND transporter AcrB in AcrAB-TolC Efflux Pumps
Get PDFIn the last years we have assisted to the steady increase in the number of bacterial strains
showing resistance to a wide variety of unrelated drugs, a phenomenon known as Multi-Drug Resistance
(MDR). Molecular efflux pumps are among the major contributors to MDR, recognizing
and expelling several noxious compounds into the external medium. In Gram-negative bacteria, efflux
pumps of the Resistance Nodulation-cell Division (RND) superfamily are particularly relevant
for the occurrence of MDR. These machineries are composed of a homotrimeric inner membrane
drug/proton RND antiporter, a homotrimeric outer membrane factor (OMF), and an undetermined
number of Membrane Fusion Proteins (MFPs), which are essential for the stability and functionality
of the pumps.
In our study we focused on the two major efflux pumps expressed in P.aeruginosa and E.coli
/ Salmonella, MexAB-OprM and AcrAB-TolC respectively. In particular, we studied the possible
multimerization of the MFP component MexA and the effects of mutations in the AcrB distal
binding pocket.
The starting point for the first part of our study was the uncertainty about the number of MexA
proteins involved in MexAB-OprM assembly. Recent studies suggested the possibility for MexA to
assemble into multimers, i.e. dimers, trimers, etc. To investigate this possibility we studied the
effects of the G72S mutation, which is known to impair the functionality of the efflux assembly
presumably by affecting the multimerization of MexA. Extended MD simulation for wild type and
mutant monomers showed large structural differences induced by the mutation. Furthermore, during
the dimerization process (simulated with protein-protein docking) only for the wild type version we
obtained potential dimeric units. New MD simulations confirmed the stability of such units and a
convergence to a common structure. These results suggest that MexA dimerization is possible and
the dimeric units are stable, within the simulation time.
In the second part of the study we investigated the effects of G288D mutation in AcrB, identified
during recent S.typhimurim infection. The AcrB variant showed an increased resistance to
ciprofloxacin, suggesting a key role of the mutation in conferring the MDR. To study how the mutation,
which is located in the distal binding pocket, might increase the efflux of ciprofloxacin we
modeled AcrB structures for the wild type and the G288D variant. Reduced models, with only
the periplasmic component, were then studied with MD simulations. The study revealed that the
G288D substitution induced local structural changes in the AcrB distal binding pocket leading to a
less compact structure with respect to the wild type. Furthermore, the mutation increased the polarity
of this region suggesting different binding mechanisms that lead to a decreased susceptibility
to ciprofloxacin and other fluoroquinolones