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Quantitative serum neopterin determination in dogs affected by Leishmaniasis
No full textLeishmaniasis is a parasitic disease caused by Leishmania spp. Once transmitted to the host, the parasite, being an obligate intracellular pathogen, localizes primarily inside macrophages, where it starts replication. Interferon γ (IFN-γ) is one of the most important mediators produced by Th1 subpopulation and it is responsible for macrophages activation that could lead to parasite control. Neopterin belongs to the chemical group known as pteridines. It is synthesised also by macrophages upon stimulation with interferon-γ and is indicative of immune system activation. Measurement of N concentrations in body fluids like blood, cerebrospinal fluid or urine provides information about the cell-mediated (T helper cells type 1 – Th1) immune sistem activation in humans; assuming that, the present study aimed at investigating if N could also be considered as an indicator of a protective reaction (Th1-mediated) of the host against Leishmania, and then if it could be used clinically as prognostic factor in leishmaniotic patients or as helpful parameter in monitoring treated patients, as investigated in men. For these purposes, the study was divided into two phases independent of each other.\ud
The first phase of the study aimed to at investigating the possible correlations between sera N values and antibody titers (dosed by immunofluorescence antibody test – IFAT) in 74 dogs, both seronegative and seropositive. In the second one, 5 patients affected by leishmaniasis and in treatment for it, were followed for a period of 24 months comparing N values to other parameters (e.g. IFAT, PCRs, citology on fine needle aspiration (FNA) of lymphnodes).\ud
Even in the absence of a statistically significant correlation between N values and IFAT titers, serum N appeared to be an interesting marker possibly able to provide an indirect value of Th1/Th2 ratio in leishmaniotic patients. It should also be further investigated its possible role in the early detection of ill patients possibly predisposed to develop renal failure and/or to relapse
Synthesis and biological activity of neuropeptide S receptor ligands\ud
No full textNeuropeptide S is a neurotransmitter constituted by 20 amino acids, which is involved in the modulation of anxiety, arousal, food intake, and insomnia. NPS activates a dehorphanized G protein-coupled receptor (NPSR) which is distributed in many brain areas with high expression in cortex, hypothalamus, amygdale, and multiple midline thalamic nuclei. Many of these areas have been functionally associated with arousal and processing of emotional\ud
behaviour and, on this basis, NPSR ligands could have therapeutic potential.\ud
While in the past years there was a big effort in the synthesis of modified peptide ligands, limited information are available about non peptide NPSR ligands. First a series of bicyclic piperazine derivatives has been published in a patent. Those compounds had been partially\ud
characterized, hence the aim of my work was the synthesis of such derivatives bearing different substituents, to define the structural figures relevant for antagonist activity.\ud
The piperazine derivatives were obtained by synthesizing the scaffold 1,1-diphenyltetrahydro-1H-oxazolo[3,4-a]pyrazin-3(5H)-one and reacting it with the respective\ud
isocyanates, acids, or pyridine acetamide analogues, to give the bicyclic piperazine derivatives (1 a-c) bearing different substituents in 7-position [...]. Furthermore, a\ud
series of modified analogues lacking the di-phenyl substitution in 1 position have been synthesized by reaction of the tetrahydro-1H-oxazolo[3,4-a]pyrazin-3(5H)-one with the suitable benzyl halide, anhydride or acyl chloride [...].\ud
The in vitro pharmacological activity of non peptide compounds was investigated in a calcium mobilization assay performed using HEK293 cells expressing the recombinant\ud
mouse NPS receptor (HEK293mNPSR).\ud
The effects of the compounds were evaluated in agonist type experiments in comparisonwith NPS, which causes a concentration dependent intracellular calcium increase. All the compound under investigation were not able to stimulate calcium mobilization up to 10 μM, hence they are not devoted of any agonistic activity.\ud
The synthesized derivatives were also tested in antagonist type experiments by evaluating the inhibition of 30 nM NPS stimulatory effect. Tested bicyclic piperazine compounds\ud
showed to inhibit NPSR with KB activity in the nanomolar range in a concentration dependent manner.\ud
Furthermore, looking at a new scaffold for the development of novel ligands, several compounds of our library, with structural feature in common with the pyperazine compound\ud
series, have been tested on mNPSR. On the base of the biological data obtained with this library, and of studies performed with molecular modeling a new series of purine derivatives were designed.\ud
The synthesis of purine analogues bearing a benzhydryl group in C-8 or N-9 position, was obtained by cyclization of the suitable 4,5-diamine-6-chloropyrimidine derivatives. The final compounds were then obtained by displacement of the 6-chloro atom with the suitable amines [...]. By reaction of the 6-amino-8-benzhydryl-9-methylpurine with\ud
different isocyanates or acids, the 6-ureido or amide purine derivatives 5 [...] have been obtained.\ud
All the new compounds were tested in the described agonist and antagonist type experiments and the results showed that only the 8-benzhydryl derivatives bearing a benzylamine group at C-6 position and a hydrogen or methyl group at N-9 position slightly inhibited the effect of NPS [...]\ud
In conclusion, none of the tested compounds were able to activate the receptor, and some of them were able to inhibit calcium mobilization after activation of the receptor by NPS.\ud
Moreover, it has been demonstrated that the CH2 linker in the side chain of benzylic piperazine derivatives is not crucial for the potency, so giving some possibility for the design of new NPSR antagonists endowed with good affinity.\ud
The purine derivatives bearing the benzhydryl group in 8-position and a benzylamine group in 6-position have been shown to slightly inhibit the NPS stimulatory effect
Investigation of the electrochemical behavior at low temperature and fast charge/discharge rate of anodic materials for lithium-ion batteries
No full textNowadays lithium-ion batteries represent the state-of-the-art of energy storage devices. Within this context the work herein presented deals with the investigation and the improvement of the electrochemical behavior of anode materials for lithium-ion batteries.\ud
Conductive agents play a fundamental role in determining the electrochemical performance of electrodes for lithium-ion batteries. Super-P carbon is often used in the\ud
electrode formulations in order to increase the electrical conductivity and to improve the contact between particles. In this work we synthesized a copper-modified Super-Pbased\ud
conductive agent, in which copper nanoparticles supported on the carbon matrix have been obtained by a microwave-assisted reduction of Cu (II) ions. The obtained\ud
copper/Super-P composite showed good dispersion of the nanoscaled copper particles on the carbon matrix. The synthesized conductive agent has been used instead of\ud
pristine Super-P during the manufacturing process of graphite and Li4Ti5O12 electrodes.\ud
Graphite is at the moment the most commonly used anode material in lithium-ion batteries. Unfortunately when graphite anodes are forced to work at low temperature\ud
and/or at high charge/discharge rate, the electrochemical performance drastically decreases. In this work electrodes made of oxidized KS-15 graphite (Timcal) have been tested at low temperature and high charge/discharge rate. Two different anodic formulations have been tested. Unmodified electrodes have been prepared using pristine Super-P as conductive agent. On the other hand, copper-modified ones have been manufactured with the synthesized copper/Super-P composite. It has been demonstrated that the use of the copper/Super-P composite in the electrode formulation leads to improved performances at low temperature (down to -30°C) and at increased charge/discharge rate. Furthermore, we propose a detailed study of the differential capacity curves dQ dE-1 vs. V and a set of experiments to investigate the electrical properties of the tested samples. The obtained results showed decreased polarization\ud
and increased electrical conductivity for copper-modified electrodes. The effect of both pristine Super-P and Cu/Super-P on anodes electrochemical behavior is explained by DC- and AC -electrical studies. The results show decreased electrical resistance and metallic-like behavior of the electronic conductivity as a function of the temperature on samples containing Cu/Super-P. It implies that when the temperature is decreased, the electrical conductivity linearly increases. Conversely, the sample containing pure Super-P follows a classical semi-conductor behavior, with the electrical conductivity decreasing with the decreasing temperature.\ud
Li4Ti5O12 is considered a safer alternative to carbonaceous anodes. The main disadvantages of LTO-based electrodes are related to the poor ionic and electronic conductivity of this compound, which determine poor rate capability and poor\ud
electrochemical performances at low-temperature. In this thesis we demonstrate that the use of the copper/Super-P composite instead of pristine Super-P positively affects the\ud
electrochemical behavior of the tested anodes. Electrodes manufactured starting from commercial LTO purchased from Aldrich, have been tested in the temperature range of\ud
-30≤ T≤ 20°C. The results demonstrate excellent performances of the copper-modified electrodes. Moreover, the rate capability and the cycling stability of the LTO electrodes have been remarkably improved by the use of copper/Super-P in the electrode composition.\ud
Rutile-TiO2 has been considered for a long time as almost inactive towards lithium insertion. Lately it has been proven that by controlling the particles size during its\ud
synthesis it is possible to reversibly insert a relevant amount of lithium ions into the rutile structure. In this work, nanoscaled rutile-TiO2 has been synthesized following a sol-gel procedure. The electrochemical tests performed on rutile electrode demonstrated the excellent electrochemical behavior at low temperature. Moreover, it has been\ud
demonstrated that rutile-TiO2 electrodes can be cycled within an enlarged potential window (0.1-3 Volts) in addition to the classical 1-3 Volts window of TiO2 system.\ud
The results obtained in this thesis have been the subject of the following publications and proceedings of congress:\ud
M. Marinaro, M. Pfanzelt, P. Kubiak, R. Marassi, M. Wohlfahrt-Mehrens, Journal of Power Sources, 196 (2011) 9825.\ud
M. Marinaro, M. Pfanzelt, P. Kubiak, R. Marassi, M. Wohlfahrt-Mehrens, 219th ECS meeting Montreal, QC, Canada 1-6. May 2011 Abs. 641.\ud
The results reported in this thesis about the electrochemical behavior of graphite and LTO electrodes will be subject of further communications
Etica d'impresa: la responsabilita' etico sociale dell'imprenditore ed i codici di condotta
Get PDFIl lavoro si propone l'obiettivo di analizzare le relazioni tra mercato ed etica, con particolare riguardo all'incidenza di scelte ispirate a valori solidaristici e personalistici nell'ambito dell'attivita' di impresa. La\ud
moderna scienza economica e giuridica, nonostante si sia sviluppata senza un costante riferimento formale al ruolo dell'etica e della morale nell'organizzazione dell'impresa, non ha rinunciato a richiamare concetti metaeconomici, quali principi e valori sociali, nella ricostruzione dei\ud
meccanismi che dovrebbero incidere sul governo della stessa.\ud
Cio' impone di superare i tradizionali strumenti di misura e previsione delle performances aziendali incentrati su parametri meramente economici. L'impresa allarga il suo raggio di azione e sotto certi aspetti deve farsi carico dei problemi sociali della comunita' in cui svolge la sua\ud
attivita'. In questa prospettiva la responsabilita' d’impresa coinvolge ed attiene anche alla responsabilita' sociale, la quale diviene obiettivo rilevante nel sistema-impresa e modo d'essere dell'azione gestionale.\ud
La responsabilita' sociale dell'impresa coinvolge non soltanto la comunita' locale, ma un ampio ventaglio di soggetti: partners commerciali, fornitori, clienti, azionisti, lavoratori dipendenti, cioe' il mercato\ud
complessivamente inteso quale "statuto normativo". Ne consegue che una indagine attenta, da un lato, ai rapporti tra autonomia negoziale, attivita' di controllo e responsabilita' nella contrattazione d'impresa e, dall'altro, al rapporto tra impresa e ambiente, consentira' di valutare l'attitudine di scelte etiche non soltanto a produrre vantaggi economici ma anche utilita' sociale. Il tutto in una prospettiva nella quale la regolamentazione del\ud
mercato non si pone in conflitto con l'iniziativa privata, bensi' contribuisce al pieno sviluppo di questa.\ud
Il fenomeno della globalizzazione ha determinato la crisi della tradizionale concezione di ordinamento giuridico e delle fonti di produzione delle norme, e ha imposto allo stesso tempo i codici di condotta fra i nuovi strumenti di creazione delle regole.\ud
Il lavoro si propone, inoltre, di esaminare tali codici di condotta nella loro componente autodisciplinare, in particolare le esperienze piu' autorevoli e consolidate dell’autodisciplina nazionale e transnazionale.\ud
Nel quinto capitolo, infine, particolare attenzione e' stata dedicata alla problematica della qualificazione giuridica dei codici di condotta.\ud
\ud
This contribution is proposed the objective to analyze the relationship between market and ethics, with particular referring to the incidence of choices inspired to solidaristic and personal values in the activity of\ud
enterprise. The modern economic and juridical science, despite are developed without a constant formal reference to the role of the ethics in the organization of the enterprise, it doesn't have renounced to use economic concepts, as principles and social values, in the reconstruction of the mechanisms that they would have to engrave on its government.\ud
This imposes to overcome the traditional tools of measure and forecast to business performanceses merely incentratis on economic parameters.\ud
The enterprise widens its ray of action and it has to confront the social problems of the community in which develops its activity. In this perspective the responsibility of enterprise involves and also concerns to\ud
the social responsibility, which becomes objective remarkable in the system-enterprise and way to be of the managerial action.\ud
The social responsibility of the enterprise involves only not the local community but many subjects: commercial partners, suppliers, customers, shareholders, dependent workers, that is the understood market altogether what normative statute. A careful investigation, from a side, to the relationships between autonomy negotiate, activity of control and responsibility in the bargaining of enterprise and, from the other, to the relationship between enterprise and environment, it will allow of to appraise only not the attitude of ethical choices to produce economic advantages but also social utility. The all in a perspective in which the regulation of the market is not set in conflict with the private initiative, on the contrary it contributes to the full development of this.\ud
The Globalization caused the crisis of the traditional concept of legal system and legal source production, and set at the same time the codes of conduct as a new rule–making instrument.\ud
This contribution also will examine the self-regulation aspects of such conduct codes, in particular the most authoritative and well-established experiences expressed by self-regulation at a national and transnational level.\ud
Finally, in the fifth chapter special attention was dedicated to the issue of judicial qualification of all conduct codes
La ricostruzione paleoclimatico-ambientale dell’Appennino umbro-marchigiano, durante il Pleistocene Medio e Superiore,\ud attraverso lo studio dei depositi in Grotta
No full textAmong the natural archives were formed in continental environments, have a prominent place the concretions in caves (speleothems) and fine sediments. In fact, the karst caves are traps sediments as paleoenvironmental information is generally stored in the memory of what happens around them, including the external system, where processes of erosion and alteration, generally, they suppress any evidence of previous environmental evolution.\ud
In recent decades, the research related to the study of the physico-chemical properties of concretional deposits (speleothems), or the fine sediments in the cave, was consequently a considerable acceleration in particular with regard to the paleoclimatic and paleoenvironmental reconstructions for the Quaternary.\ud
The caves represent, then, very important sites of research of the fossil plant that, trapped in sediments, in calcite or/and gypsum concretions, allow to palinologist and geologist to discover plant change through geological ages and, then, the climate variations during the Quaternary age.\ud
The goal of this thesis is the study of a caves in the umbro-marchean Apennines (Marche region, central Italy) and, in particular, the two karts complexes: karst Complex of Frasassi and karst Complex of Caprelle.\ud
This research has been led on two fronts strictly interrelated, one of geomorphological/geochronological study and the second of palinological analysis. The second allowed the paleoclimatic and paleoflora-vegetation\ud
reconstruction when the two karst complexes were going to be formed and gave a contribute to chronological placement of deposits.\ud
This research began by collecting samples in Frasassi Complex (10 samples belonging to different karst levels and in different caves of the wide Complex) and in Caprelle Complex (9 samples collected at different sites and\ud
depth, down to about 75 metres). Sampled sediments have been treated for pollen extraction (this method provides deflocculation by Na-pyrophosphate 10%, solution of carbonates by HCl 10%, acetolysis by Erdtman 1960, enrichment with heavy liquid Na-metatungstate hydrate, solution of silicates by HF 40%), in the Paleobotany and Pollen Analysis Laboratory of Modena and Reggio Emilia\ud
University. The results gave an enough pollen quantity, from some studied samples, and the data are consistent with the typology of substrates usually studied in speleo-palinology. Collected pollen, as expected, had a good/excellent state of conservation, so to allow an easier identification.\ud
Through the study of these two karst complexes, this research intends to give a contribute to the knowledge of environmental elements, to the reconstruction of paleoenvironmental and paleoclimatic of the karst area in the umbro-marchean area, in some cases obtaining documents handy to understanding cave’s and deposits genesis, during the karst complex evolution.\ud
The whole umbro-marchean Apennines is constituted by sedimentary rocks (CALAMITA & PIERANTONI, 1993; DEIANA et al., 2002). Also on the ground are well clear the effects of the deforming and compressive tectonic phases,\ud
happened during the Neogene, and of extensional ones happened during the Quaternary. During this last period, at last, most evident alterations of landscape result caused by surface and depth karst processes with genesis of caves\ud
(speleogenesis).\ud
Such researches were begun in bogs, swamps and ponds, in the sixties and carried on till nowdays, enriched since the eighties by palinological analysis conducted in hypogeum environments. Deposits of Frasassi e Caprelle have given\ud
an important contribute to paleoenvironmental reconstruction of Pleistocene in Italy and particularly in Central Italy. Various past and recent italian sequences\ud
have been analysed, based on pollen data quality and an adeguate chronostratigraphy reference frame, and were indispensable for doing a comparison with palinological records of karst complexes of Frasassi and Caprelle.\ud
In the study of cave sediments is very important to pay attention to the site where collect samples, in order to grant the possibility to find pollen in good quantity and so have the possibility to interrelate palinological analysis and geomorphological and geochronological data (absolute and relative chronologies).\ud
In Italy, only eight cave sites were studied by a palinological point of view. In some of these sites, as Grotta di Salomone and Sant’Angelo (Abruzzo region),\ud
concretions were studied, whereas on other caves palinological analysis were focused depth deposits. In Buco Bucone cave, in Grotta di Valle delle Vacche cave, in the Caprelle and Frasassi complexes sample sites were not at the surface but at a depth of 150 metres. Before this thesis, in Central Italy only three caves have been analysed by a palinological point of view
Effect of Malaria-HIV coinfections on innate dendritic cells phenotypic functions: immunomodulations of neonatal responses to malaria blood stage vaccine candidates
No full textIn this thesis we endeavored to explain how malaria impacts on innate immune dendritic cells in both the adult and cord blood and how this impacts on the HIV transmission during confections. Additionally, the effect of maternal single malaria and HIV infections or malaria/HIV coinfections on neonate dendritic cells functions and their responses to malaria vaccine candidates was investigated. \ud
We report that cord blood is intrinsically immature (more CD209, less CD1a), develop higher activation (HLA-DR) and has inferior cytokine production, characterized by less pro-inflammatory IL-12 and higher inhibitory IL-10 production than the adult blood when exposed to both malaria and HIV infectious agents. During a comparison of cord blood from malaria exposed and naïve individuals, the cord blood DCs from Rome consistently showed higher potential of maturation via increased CD1a and C83 expression than the all (infected or uninfected) malaria exposed samples from Yaoundé. Furthermore active malaria and/or HIV infection in pregnancy significantly lowered co-stimulatory CD80 and CD86 expression in neonate DCs than in either malaria naïve or malaria negatives during pregnancy. Cord blood T cells from HIV or infected malaria/HIV co-infected mothers had a decreased IL17a production during mixed lymphocyte reactions (MLRs) as compared to malaria/ non-infected pregnant women (p<0.01). Similarly the cord blood from the same individuals had significantly lower CD4 T cell proliferation rates. Interestingly incubation with malaria vaccine antigens Pp-AMA-1resulted in significant higher production of IL17a than when incubated with Ec-MSP-142(P<0.05) and medium (p<0.01) under all cohorts. Additionally, incubation with Pp-AMA-1led to comparable expansion rates with those in PHA (p>0.05) but higher expansion than those in Ec-MSP-142 or medium (p<0.05). \ud
We conclude that cord blood DCs have intrinsically inferior maturation and cytokine production ability in response to malaria and/or HIV infection than adults, thus these differences should be considered duringvaccine design and evaluation for use in infants. Maternal malaria affected the CBDC phenotypes while coinfections with HIV additionally dysregulated their functions via preferential loss of IL17a production and poor CD4 T cell expansion, indicating that these two can be biomarkers that may be useful to study the immunomodulation and pathogenesis in malaria/HIV dual infections. Interestingly, there were increased functional phenotype responses by cord blood DCs from both HIV and malaria infected mothers when incubated with malaria vaccine antigen Pp-AMA-1 and not Ec-MSP-142. This suggests that Pp-AMA-1stimulates innate immunity and may revert P. falciparum dysregulation of DC and subsequent adaptive protection, thus a better understanding of the mechanisms involved might be useful for development of improved malaria pediatric vaccine especially during dual infection with HIV.\u
Interactions between Asaia, Plasmodium and Anopheles: new insights in mosquito symbiosis and implications in Malaria Symbiotic Control
No full textThe recent identification of acetic acid bacteria belonging to the genus Asaia, that are stably associated with larvae and adults of different species of malaria transmitting mosquitoes, in particular Anopheles stephensi, indicates this bacterium as a suitable candidate for the paratransgenic or symbiotic control of the malaria infection. The presence of Asaia in the mosquito reproductive system represents an additional intriguing feature, particularly important towards the development of Paratransgenetically Modified (PM) mosquitoes that can mix with natural populations. By the means of Real Time quantitative PCR I have investigated the relative amount of Asaia and Plasmodium in co-infected mosquitoes thus providing evidence that the malaria infection does not interfere with the blooming of Asaia that follows a blood meal. Furthermore, by immunological studies we provided evidences that Asaia induces ‘in vitro’ the expression of antimicrobial peptides (AMPs) in the mosquito and that, the adaptation of Asaia to the life into the insect, doesn’t appear to be related with a reduced immunogenicity, but with a resistance to the immune reaction. In addition, by fluorescent recombinant strains of both Asaia and Plasmodium, we were able to precisely define the co-localisation of these organisms at the level of mosquito midgut and salivary glands. It was also considered necessary to verify the Asaia role about the vitality of adults mosquitoes. For this aim, has been adopted a novel strategy using monoclonal antibodies against Asaia, to interfere with the function of these bacteria by antibody opsonization. \ud
These studies while confirming the potential suitable use of Asaia as microbial agent for the paratransgenic control of malaria infection. \ud
Beyond bacteria, yeast-like organisms were also present in the mosquito midgut and in reproductive systems. We have identified Wickerhamomyces anomalus (Saccharomycetales) as an important component of the yeast microbiome in this mosquito species, suggesting multiple transmission patterns. The possibility to easily manipulate yeast, and the chance to express effector molecules in a eukaryotic organism renders W. anomalus another good potential candidate for paratransgenesis.\u
Cis-acting and trans-acting factors regulating gene expression during cold stress in Eschirichia coli
No full textDevelopment of thermo-stable, pH-sensitive colour variants of Aequorea victoria green fluorescent protein and evidence for a possible chaperone activity of translation initiation factor IF2
No full textQuesta tesi consiste di tre capitoli tra loro interconnessi, ma indipendenti, ciascuno dei quali affronta\ud
problematiche mirate ad obiettivi specifici. Il primo capitolo descrive lo sviluppo di un una variante gialla della proteina GFP di Aequorea victoria che mostra una notevole sensibilità alle variazioni di pH. Il secondo capitolo e' basato sull’idea di produrre, mediante ingegneria genetica, delle molecole di GFP destabilizzate al fine di studiare l’espressione transiente di proteine citoplasmatiche. Il terzo capitolo e' stato proposto per assemblare una serie di evidenze sperimentali riguardo l'attivita' di “chaperone” del fattore d’inizio della traduzione IF2 usando la GFP denaturata come substrato modello. In linea con il primo obiettivo, e' stata ingegnerizzata una proteina fluorescente gialla (YFP) le cui caratteristiche sono una migliorata termo stabilita' e una più efficiente cinetica di “folding”. In conseguenza di queste proprieta', tale proteina e' stata chiamata "fast folder thermostable YFP" (FFTS-YFP). Inoltre, utilizzando un simile approccio, in questo studio e' stata preparata e purificata una variante della superfolder-GFP di colore ciano che mostra notevole sensibilita' al pH ed ha dei picchi di massima emissione di fluorescenza a pH 4,8 e 3,8. A causa di tali caratteristiche, la variante proteica e' stata denominata "acid stable cyan fluorescent protein" (AcS-CFP). Per quanto riguarda il secondo obiettivo di questa tesi, sono state prodotte mediante modificazione genetica tre varianti instabili della GFP, e le loro proprieta' sono state analizzate in test in vivo. E’ stato dimostrato in tal modo che l’emivita di queste proteine mutanti, che presentano dei pentapeptidi all’ estremità C-terminale sono: GFP-104 (t ½ = 64.86 ± 3.84 h), GFP-108 (t ½ = 126 ± 5.36 h), GFP-105 (t ½ = 170 ± 0.562 h), e GFP-102 (controllo stabile, t ½ >180 h). I risultati ottenuti indicano che le modificazioni introdotte al C-terminale hanno reso tali proteine progressivamente instabili nel confronto con GFP-102, il controllo stabile di tale proteina. Per quanto riguarda il terzo obiettivo di questa tesi, e' stato dimostrato che il fattore d’inizio della traduzione batterica IF2 possiede una quantificabile attivita' “chaperonica”.\ud
IF2α, la versione intera della proteina, e' in grado di stimolare il “refolding” della proteina GFP denaturata in\ud
condizioni acide con piu' efficienza rispetto ai mutanti di delezione IF2b e IF2DN. La presenza di IF2α, infatti,\ud
permette alla GFP denaturata di recuperare un livello di “refolding” fino al 96%, mentre la stimolazione ottenuta\ud
con IF2b e IF2DN si attesta su livelli rispettivamente di 69% e 42.3%. Tali differenze indicano che una parziale o\ud
totale eliminazione della regione N-terminale di IF2 e' in grado di alterare significativamente l’attivita'\ud
“chaperone-like” di IF2. Inoltre, l’aggiunta di IF2α alle proteine DnaK, DnaJ e GrpE provoca un aumento\ud
significativo (P <0.05) delle loro capacita' di “refolding” della GFP di un valore di 26.3%, comparato ai livelli di\ud
“refolding” ottenuti dalle tre proteine in assenza di IF2. Pertanto, in questa attivita' di “refolding” della proteina\ud
GFP denaturata termicamente, IF2 mostra un’attivita' sinergica con le principali proteine ad attivita'\ud
“chaperonica”. In conclusione, i risultati di questo lavoro mostrano in maniera evidente che i principali obiettivi\ud
dichiarati in ciascuna delle tre sezioni sono stati regolarmente ottenuti.\ud
\ud
This thesis consists of three inter-related, independent chapters, each one dealing with highly focused objectives. The first chapter describes the development of a thermo-stable yellow variant of Aequorea victoria green fluorescent protein (GFP) displaying a remarkable sensitivity to pH changes; the second one was launched with the purpose of producing, by genetic engineering, destabilized GFPs having potential for studying transient expression of cytoplasmic proteins; and the third chapter was proposed to pileup evidences for chaperone activity of bacterial initiation factor IF2 using denatured GFP as a model substrate.\ud
Accordingly, in line with the first objective, a yellow fluorescent protein (YFP) variant with improved thermostability and folding kinetics was engineered and because of these properties it was named “fast folder thermostable YFP” (FFTS-YFP). In addition, in this study it was prepared and purified a pHsensitive cyan variant of superfolder GFP, which displays preference for acidic conditions and the highest fluorescence emission at pH 4.8 and 3.8. Because of such characteristics, the variant was named “acid stable cyan fluorescent protein” (AcS-CFP). Regarding the second objective of this thesis, three unstable GFP variants were engineered and their properties were evaluated in vivo. It was found that the in vivo halflives of these mutant proteins carrying pentapetides at the C-terminus were: GFP-104 (t ½ = 64.86 ± 3.84 h), GFP-105 (t ½ = 170 ± 0.562 h), GFP-108 (t ½ = 126 ± 5.36 h) and GFP-102 (stable control, t ½ >180 h).\ud
The results indicate that the C-terminal tagging has rendered the engineered proteins progressively unstable\ud
as compared to the stable control, GFP102. As for the third major objective of this work, our data show that IF2 can indeed promote GFP refolding and that the chaperone activity of IF2α on the refolding of aciddenatured GFP is significantly (P < 0.05) more pronounced (96%) than that of IF2β (69%) and IFN (42.3%). Therefore, the absence of IF2N1 (as in IF2β) reduces the chaperone activity of IF2 by 30% and the total loss of the IF2 NTD (i.e. N1 + N2) as in IF2N causes a 57% activity reduction. These results\ud
indicate that the N-terminal domain of IF2 plays a major role in the chaperone activity of the factor. This is the first time that IF2α and IF2β prove to have a significant activity difference. Addition of IF2α to DnaK, DnaJ and GrpE was found to increase significantly (P < 0.05) by 26.26% the combined activity of these chaperones indicating that there is indeed a strong synergy between IF2 and the known chaperones.\ud
Overall, the present findings are instrumental in redefining the functional role of IF2 which cannot be regarded as being strictly restricted to its well documented functions in bacterial translation initiation. In\ud
conclusion, the results clearly show that the main objectives, initially stated under each chapter, were\ud
achieved
