Expression of the repressor element-1 silencing transcription factor (REST) is regulated by IGF-I and PKC in human neuroblastoma cells

The repressor element 1-silencing transcription factor (REST) was first identified as a protein that binds to a 21-bp DNA sequence element (known as repressor element 1 (RE1)) resulting in transcriptional repression of the neural-specific genes [Chong et al., 1995; Schoenherr and Anderson, 1995]. The original proposed role for REST was that of a factor responsible for restricting neuronal gene expression to the nervous system by silencing expression of these genes in non-neuronal cells. Although it was initially thought to repress neuronal genes in non-neuronal cells, the role of REST is complex and tissue dependent. In this study I investigated any role played by REST in the induction and patterning of differentiation of SH-SY5Y human neuroblastoma cells exposed to IGF-I. and phorbol 12- myristate 13-acetate (PMA) To down-regulate REST expression we developed an antisense (AS) strategy based on the use of phosphorothioate oligonucleotides (ODNs). In order to evaluate REST mRNA levels, we developed a real-time PCR technique and REST protein levels were evaluated by western blotting. Results showed that nuclear REST is increased in SH-SY5Y neuroblastoma cells cultured in SFM and exposed to IGF-I for 2-days and it then declines in 5-day-treated cells concomitant with a progressive neurite extension. Also the phorbol ester PMA was able to increase nuclear REST levels after 3-days treatment concomitant to neuronal differentiation of neuroblastoma cells, whereas, at later stages, it is down-regulated. Supporting these data, the exposure to PKC inhibitors (GF10923X and Gö6976) and PMA (16nM) reverted the effects observed with PMA alone. REST levels were related to morphological differentiation, expression of growth coneassociated protein 43 (GAP-43; a gene not regulated by REST) and of synapsin I and βIII tubulin (genes regulated by REST), proteins involved in the early stage of neuronal development. We observed that differentiation of SH-SY5Y cells by IGF-I and PMA was accompanied by a significant increase of these neuronal markers, an effect that was concomitant with REST decrease. In order to relate the decreased REST expression with a progressive neurite extension, I investigated any possible involvement of the ubiquitin–proteasome system (UPS), a multienzymatic pathway which degrades polyubiquinated soluble cytoplasmic proteins [Pickart and Cohen, 2004]. For this purpose, SH-SY5Y cells are concomitantly exposed to PMA and the proteasome inhibitor MG132. In SH-SY5Y exposed to PMA and MG 132, we observed an inverse pattern of expression of synapsin I and β- tubulin III, two neuronal differentiation markers regulated by REST. Their cytoplasmic levels are reduced when compared to cells exposed to PMA alone, as a consequence of the increase of REST expression by proteasome inhibitor. The majority of proteasome substrates identified to date are marked for degradation by polyubiquitinylation; however, exceptions to this principle, are well documented [Hoyt and Coffino, 2004]. Interestingly, REST degradation seems to be completely ubiquitin-independent. The expression pattern of REST could be consistent with the theory that, during early neuronal differentiation induced by IGF-I and PKC, it may help to repress the expression of several genes not yet required by the differentiation program and then it declines later. Interestingly, the observation that REST expression is progressively reduced in parallel with cell proliferation seems to indicate that the role of this transcription factor could also be related to cell survival or to counteract apotosis events [Lawinger et al., 2000] although, as shown by AS-ODN experiments, it does not seem to be directly involved in cell proliferation. Therefore, the decline of REST expression is a comparatively later event during maturation of neuroroblasts in vitro. Thus, we propose that REST is regulated by growth factors, like IGF-I, and PKC activators in a time-dependent manner: it is elevated during early steps of neural induction and could contribute to down-regulate genes not yet required by the differentiation program while it declines later for the acquisition of neural phenotypes, concomitantly with a progressive neurite extension. This later decline is regulated by the proteasome system activation in an ubiquitin-indipendent way and adds more evidences to the hypothesis that REST down-regulation contributes to differentiation and arrest of proliferation of neuroblastoma cells. Finally, the glycosylation pattern of the REST protein was analysed, moving from the observation that the molecular weight calculated on REST sequence is about 116 kDa but using western blotting this transcription factor appears to have distinct apparent molecular weight (see Table 1.1): this difference could be explained by post-translational modifications of the proteins, like glycosylation. In fact recently, several studies underlined the importance of O-glycosylation in modulating transcriptional silencing, protein phosphorylation, protein degradation by proteasome and protein–protein interactions [Julenius et al., 2005; Zachara and Hart, 2006]. Deglycosilating analysis showed that REST protein in SH-SY5Y and HEK293 cells is Oglycosylated and not N-glycosylated. Moreover, using several combination of deglycosilating enzymes it is possible to hypothesize the presence of Gal-β(1-3)-GalNAc residues on the endogenous REST, while β(1-4)-linked galactose residues may be present on recombinant REST protein expressed in HEK293 cells. However, the O-glycosylation process produces an immense multiplicity of chemical structures and monosaccharides must be sequentially hydrolyzed by a series of exoglycosidase. Further experiments are needed to characterize all the post-translational modification of the transcription factor REST.

B-type cytochromes of the DOMON domain superfamily (Novel redox elements of plant plasma membrane)

The aim of the present study is understanding the properties of a new group of redox proteins having in common a DOMON-type domain with characteristics of cytochromes b. The superfamily of proteins containing a DOMON of this type includes a few protein families. With the aim of better characterizing this new protein family, the present work addresses both a CyDOM protein (a cytochrome b561) and a protein only comprised of DOMON(AIR12), both of plant origin. Apoplastic ascorbate can be regenerated from monodehydroascorbate by a trans-plasma membrane redox system which uses cytosolic ascorbate as a reductant and comprises a high potential cytochrome b. We identified the major plasma membrane (PM) ascorbate-reducible b-type cytochrome of bean (Phaseolus vulgaris) and soybean (Glycine max) hypocotyls as orthologs of Arabidopsis auxin-responsive gene air12. The protein, which is glycosylated and glycosylphosphatidylinositol-anchored to the external side of the PM in vivo, was expressed in Pichia pastoris in a recombinant form, lacking the glycosylphosphatidylinositol-modification signal, and purified from the culture medium. Recombinant AIR12 is a soluble protein predicted to fold into a β-sandwich domain and belonging to the DOMON superfamily. It is shown to be a b-type cytochrome with a symmetrical α-band at 561 nm, to be fully reduced by ascorbate and fully oxidized by monodehydroascorbate. Redox potentiometry suggests that AIR12 binds two high-potential hemes (Em,7 +135 and +236 mV). Phylogenetic analyses reveal that the auxin-responsive genes AIR12 constitute a new family of plasma membrane b-type cytochromes specific to flowering plants. Although AIR12 is one of the few redox proteins of the PM characterized to date, the role of AIR12 in trans-PM electron transfer would imply interaction with other partners which are still to be identified. Another part of the present project was aimed at understanding of a soybean protein comprised of a DOMON fused with a well-defined b561 cytochrome domain (CyDOM). Various bioinformatic approaches show this protein to be composed of an N-terminal DOMON followed by b561 domain. The latter contains five transmembrane helices featuring highly conserved histidines, which might bind haem groups. The CyDOM has been cloned and expressed in the yeast Pichia pastoris, and spectroscopic analyses have been accomplished on solubilized yeast membranes. CyDOM clearly reveal the properties of b-type cytochrome. The results highlight the fact that CyDOM is clearly able to lead an electron flux through the plasmamembrane. Voltage clamp experiments demonstrate that Xenopus laevis oocytes transformed with CyDOM of soybean exhibit negative electrical currents in presence of an external electron acceptor. Analogous investigations were carried out with SDR2, a CyDOM of Drosophila melanogaster which shows an electron transport capacity even higher than plant CyDOM. As quoted above, these data reinforce those obtained in plant CyDOM on the one hand, and on the other hand allow to attribute to SDR2-like proteins the properties assigned to CyDOM. Was expressed in Regenerated tobacco roots, transiently transformed with infected a with chimeral construct GFP: CyDOM (by A. rhizogenes infection) reveals a plasmamembrane localization of CyDOM both in epidermal cells of the elongation zone of roots and in root hairs. In conclusion. Although the data presented here await to be expanded and in part clarified, it is safe to say they open a new perspective about the role of this group of proteins. The biological relevance of the functional and physiological implications of DOMON redox domains seems noteworthy, and it can but increase with future advances in research. Beyond the very finding, however interesting in itself, of DOMON domains as extracellular cytochromes, the present study testifies to the fact that cytochrome proteins containing DOMON domains of the type of “CyDOM” can transfer electrons through membranes and may represent the most important redox component of the plasmamembrane as yet discovered.

Molecular characterization of the human gut microbiota: the effect of aging

Age-related physiological changes in the gastrointestinal tract, as well as modification in lifestyle, nutritional behaviour, and functionality of the host immune system, inevitably affect the gut microbiota. The study presented here is focused on the application and comparison of two different microarray approaches for the characterization of the human gut microbiota, the HITChip and the HTF-Microb.Array, with particular attention to the effects of the aging process on the composition of this ecosystem. By using the Human Intestinal Tract Chip (HITChip), recently developed at the Wageningen University, The Netherland, we explored the age-related changes of gut microbiota during the whole adult lifespan, from young adults, through elderly to centenarians. We observed that the microbial composition and diversity of the gut ecosystem of young adults and seventy-years old people is highly similar but differs significantly from that of the centenarians. After 100 years of symbiotic association with the human host, the microbiota is characterized by a rearrangement in the Firmicutes population and an enrichment of facultative anaerobes. The presence of such a compromised microbiota in the centenarians is associated with an increased inflammation status, also known as inflamm-aging, as determined by a range of peripheral blood inflammatory markers. In parallel, we overtook the development of our own phylogenetic microarray with a lower number of targets, aiming the description of the human gut microbiota structure at high taxonomic level. The resulting chip was called High Taxonomic level Fingerprinting Microbiota Array (HTF-Microb.Array), and was based on the Ligase Detection Reaction (LDR) technology, which allowed us to develop a fast and sensitive tool for the fingerprint of the human gut microbiota in terms of presence/absence of the principal groups. The validation on artificial DNA mixes, as well as the pilot study involving eight healthy young adults, demonstrated that the HTF-Microb.Array can be used to successfully characterize the human gut microbiota, allowing us to obtain results which are in approximate accordance with the most recent characterizations. Conversely, the evaluation of the relative abundance of the target groups on the bases of the relative fluorescence intensity probes response still has some hindrances, as demonstrated by comparing the HTF.Microb.Array and HITChip high taxonomic level fingerprints of the same centenarians.

Quali-quantitative study of the phenolic and polyphenolic compounds and their antioxidant capacity in vegetal matrix processed with different technologies

Food technologies today mean reducing agricultural food waste, improvement of food security, enhancement of food sensory properties, enlargement of food market and food economies. Food technologists must be high-skilled technicians with good scientific knowledge of food hygiene, food chemistry, industrial technologies and food engineering, sensory evaluation experience and analytical chemistry. Their role is to apply the modern vision of science in the field of human nutrition, rising up knowledge in food science. The present PhD project starts with the aim of studying and improving frozen fruits quality. Freezing process in very powerful in preserve initial raw material characteristics, but pre-treatment before the freezing process are necessary to improve quality, in particular to improve texture and enzymatic activity of frozen foods. Osmotic Dehydration (OD) and Vacuum Impregnation (VI), are useful techniques to modify fruits and vegetables composition and prepare them to freezing process. These techniques permit to introduce cryo-protective agent into the food matrices, without significant changes of the original structure, but cause a slight leaching of important intrinsic compounds. Phenolic and polyphenolic compounds for example in apples and nectarines treated with hypertonic solutions are slightly decreased, but the effect of concentration due to water removal driven out from the osmotic gradient, cause a final content of phenolic compounds similar to that of the raw material. In many experiment, a very important change in fruit composition regard the aroma profile. This occur in strawberries osmo-dehydrated under vacuum condition or under atmospheric pressure condition. The increment of some volatiles, probably due to fermentative metabolism induced by the osmotic stress of hypertonic treatment, induce a sensory profile modification of frozen fruits, that in some way result in a better acceptability of consumer, that prefer treated frozen fruits to untreated frozen fruits. Among different processes used, a very interesting result was obtained with the application of a osmotic pre-treatment driven out at refrigerated temperature for long time. The final quality of frozen strawberries was very high and a peculiar increment of phenolic profile was detected. This interesting phenomenon was probably due to induction of phenolic biological synthesis (for example as reaction to osmotic stress), or to hydrolysis of polymeric phenolic compounds. Aside this investigation in the cryo-stabilization and dehydrofreezing of fruits, deeper investigation in VI techniques were carried out, as studies of changes in vacuum impregnated prickly pear texture, and in use of VI and ultrasound (US) in aroma enrichment of fruit pieces. Moreover, to develop sensory evaluation tools and analytical chemistry determination (of volatiles and phenolic compounds), some researches were bring off and published in these fields. Specifically dealing with off-flavour development during storage of boiled potato, and capillary zonal electrophoresis (CZE) and high performance liquid chromatography (HPLC) determination of phenolic compounds.

Characterization of the Staphylococcus aureus bone sialoprotein-binding protein SdrE and the serine protease EpiP

In an attempt to develop a Staphylococcus aureus vaccine, we have applied reverse vaccinology approach, mainly based on in silico screening and proteomics. By using this approach SdrE, a protein belonging to serine-aspartate repeat protein family was identified as potential vaccine antigen against S. aureus. We have investigated the biochemical properties as well as the vaccine potential of SdrE and its highly conserved CnaBE3 domain. We found the protein SdrE to be resistant to trypsin. Further analysis of the resistant fragment revealed that it comprises a CnaBE3 domain, which also showed partial trypsin resistant behavior. Furthermore, intact mass spectrometry of rCnaBE3 suggested the possible presence of isopeptide bond or some other post-translational modification in the protein.However, this observation needs further investigation. Differential Scanning Fluorimetry study reveals that calcium play role in protein folding and provides stability to SdrE. At the end we have demonstrated that SdrE is immunogenic against clinical strain of S. aureus in murine abscess model. In the second part, I characterized a protein, annotated as epidermin leader peptide processing serine protease (EpiP), as a novel S. aureus vaccine candidate. The crystal structure of the rEpiP was solved at 2.05 Å resolution by x-ray crystallography . The structure showed that rEpiP was cleaved somewhere between residues 95 and 100 and cleavage occurs through an autocatalytic intra-molecular mechanism. In addition, the protein expressed by S. aureus cells also appeared to undergo a similar processing event. To determine if the protein acts as a serine protease, we mutated the catalytic serine 393 residue to alanine, generating rEpiP-S393A and solved its crystal structure at a resolution of 1.95 Å. rEpiP-S393A was impaired in its protease activity, as expected. Protective efficacy of rEpiP and the non-cleaving mutant protein was comparable, implying that the two forms are interchangeable for vaccination purposes.

Use of essential oils and biocontrol cultures for the improvement of shelf-life of fresh cut products

The demand of minimally processed fruits and vegetables has increased in the last years. However, their intrinsic characteristics may favor the growth of pathogens and spoilage microbiota. The negative effects on human health reported for some traditional chemical sanitizers have justified the search for substitutes to guarantee food safety and quality. In this work we have evaluate the potential of some essential oils and their components to improve the safety and the shelf life of Lamb’s lettuce (Valerianella locusta) and apples (Golden delicious). Moreover, the effects of selected lactic acid bacteria alone or in combination with essential oils or their components, on the shelf-life and safety as well as organoleptic properties of minimally processed products, were evaluated. Since the lack of knowledge of microbial cell targets of essential oils represent one of the most important limit to the use of these molecules at industrial level, another aim of this thesis was the study of the action mechanisms of essential oils and their components. The results obtained showed the beneficial effects of the natural antimicrobials as well as the selected lactic acid bacteria on minimally processed fruit and vegetable safety and shelf-life, without detrimental effects on the quality parameters. The beneficial effects obtained by the use of the selected biocontrol agents were further increased combining them with selected natural antimicrobials. The natural antimicrobial employed induced noticeable modifications of membrane fatty acid profiles and volatile compounds produced by microbial cells during the growth. The modification of the expression in genes involved in fatty acid biosynthesis suggesting that the cytoplasmic membrane of microbial cells is one of the major cellular target of essential oils and their components. The comprehension of microbial stress response mechanisms can contribute to the scaling up of natural antimicrobials and bio-control agents at industrial level.

Synthesis and surface modification of silver and gold nanoparticles. Nanomedicine applications against Glioblastoma Multiforme.

In the last decades noble metal nanoparticles (NPs) arose as one of the most powerful tools for applications in nanomedicine field and cancer treatment. Glioblastoma multiforme (GBM), in particular, is one of the most aggressive malignant brain tumors that nowadays still presents a dramatic scenario concerning median survival. Gold nanorods (GNRs) and silver nanoparticles (AgNPs) could find applications such as diagnostic imaging, hyperthermia and glioblastoma therapy. During these three years, both GNRs and AgNPs were synthesized with the “salt reduction” method and, through a novel double phase transfer process, using specifically designed thiol-based ligands, lipophilic GNRs and AgNPs were obtained and separately entrapped into biocompatible and biodegradable PEG-based polymeric nanoparticles (PNPs) suitable for drug delivery within the body. Moreover, a synergistic effect of AgNPs with the Alisertib drug, were investigated thanks to the simultaneous entrapment of these two moieties into PNPs. In addition, Chlorotoxin (Cltx), a peptide that specifically recognize brain cancer cells, was conjugated onto the external surface of PNPs. The so-obtained novel nanosystems were evaluated for in vitro and in vivo applications against glioblastoma multiforme. In particular, for GNRs-PNPs, their safety, their suitability as optoacoustic contrast agents, their selective laser-induced cells death and finally, a high tumor retention were all demonstrated. Concerning AgNPs-PNPs, promising tumor toxicity and a strong synergistic effect with Alisertib was observed (IC50 10 nM), as well as good in vivo biodistribution, high tumor uptake and significative tumor reduction in tumor bearing mice. Finally, the two nanostructures were linked together, through an organic framework, exploiting the click chemistry azido-alkyne Huisgen cycloaddition, between two ligands previously attached to the NPs surface; this multifunctional complex nanosystem was successfully entrapped into PNPs with nanoparticles’ properties maintenance, obtaining in this way a powerful and promising tool for cancer fight and defeat.

Transcriptional dynamics of the human DMD locus

The human DMD locus encodes dystrophin protein. Absence or reduced levels of dystrophin (DMD or BMD phenotype, respectively) lead to progressive muscle wasting. Little is known about the complex coordination of dystrophin expression and its transcriptional regulation is a field of intense interest. In this work we found that DMD locus harbours multiple long non coding RNAs which orchestrate and control transcription of muscle dystrophin mRNA isoforms. These lncRNAs are tissue-specific and highly expressed during myogenesis, suggesting a possible role in tissue-specific expression of DMD gene isoforms. Their forced ectopic expression in human muscle and neuronal cells leads to a specific and negative regulation of endogenous dystrophin full lenght isoforms. An intriguing aspect regarding the transcription of the DMD locus is the gene size (2.4Mb). The mechanism that ensures the complete synthesis of the primary transcript and the coordinated splicing of 79 exons is still completely unknown. By ChIP-on-chip analyses, we discovered novel regions never been involved before in the transcription regulation of the DMD locus. Specifically, we observed enrichments for Pol II, P-Ser2, P-Ser5, Ac-H3 and 2Me-H3K4 in an intronic region of 3Kb (approximately 21Kb) downstream of the end of DMD exon 52 and in a region of 4Kb spanning the DMD exon 62. Interestingly, this latter region and the TSS of Dp71 are strongly marked by 3Me-H3K36, an histone modification associated with the regulation of splicing process. Furthermore, we also observed strong presence of open chromatin marks (Ac-H3 and 2Me-H3K4) around intron 34 and the exon 45 without presence of RNA pol II. We speculate that these two regions may exert an enhancer-like function on Dp427m promoter, although further investigations are necessary. Finally, we investigated the nuclear-cytoplasmic compartmentalization of the muscular dystrophin mRNA and, specifically, we verified whether the exon skipping therapy could influence its cellular distribution.

The Council of Ministers of the European Union After the Lisbon Treaty

The Treaty of Lisbon has brought remarkable changes and innovations to the European Union. As far as the Council of Ministers of the European Union (“the Council” hereinafter) is concerned, there are two significant innovations: double qualified majority voting and new rotating Presidency scheme, which are considered to make the working of the Council more efficiently, stably and consistently. With the modification relating to other key institutions, the Commission and the European Parliament, and with certain procedures being re-codified, the power of the Council varies accordingly, where the inter-institutional balance counts for more research. As the Council is one of the co-legislatures of the Union, the legislative function of it would be probably influenced, positively or negatively, by the internal innovations and the inter-institutional re-balance. Has the legislative function of the Council been reinforced or not? How could the Council better reach its functional goal designed by the Treaties’ drafter? How to evaluate the Council’s evolution after Lisbon Treaty in the light of European integration? This thesis is attempting to find the answers by analyzing two main internal innovations and inter-institutional re-balance thereinafter.

Electrochemical surface modification of Single walled carbon nanotubes and graphene-based electrodes for (bio)sensing applications

Sensors are devices that have shown widespread use, from the detection of gas molecules to the tracking of chemical signals in biological cells. Single walled carbon nanotube (SWCNT) and graphene based electrodes have demonstrated to be an excellent material for the development of electrochemical biosensors as they display remarkable electronic properties and the ability to act as individual nanoelectrodes, display an excellent low-dimensional charge carrier transport, and promote surface electrocatalysis. The present work aims at the preparation and investigation of electrochemically modified SWCNT and graphene-based electrodes for applications in the field of biosensors. We initially studied SWCNT films and focused on their topography and surface composition, electrical and optical properties. Parallel to SWCNTs, graphene films were investigated. Higher resistance values were obtained in comparison with nanotubes films. The electrochemical surface modification of both electrodes was investigated following two routes (i) the electrografting of aryl diazonium salts, and (ii) the electrophylic addition of 1, 3-benzodithiolylium tetrafluoroborate (BDYT). Both the qualitative and quantitative characteristics of the modified electrode surfaces were studied such as the degree of functionalization and their surface composition. The combination of Raman, X-ray photoelectron spectroscopy, atomic force microscopy, electrochemistry and other techniques, has demonstrated that selected precursors could be covalently anchored to the nanotubes and graphene-based electrode surfaces through novel carbon-carbon formation.

Study of the genes involved in Naphthenic acids (NAs) degradation by Rhodococcus spp.

Naphthenic acids (NAs) are an important group of organic pollutants mainly found in hydrocarbon deposits. Although these compounds are toxic, recalcitrant, and persistent in the environment, we are just learning the diversity of microbial communities involved in NAs- degradation and the mechanisms by which NAs are biodegraded. Studies have shown that naphthenic acids are susceptible to biodegradation, which decreases their concentration and reduces toxicity. Nevertheless, little is still known about their biodegradability. The present PhD Thesis’s work is aimed to study the biodegradation of simple model NAs using bacteria strains belonging to the Rhodococcus genus. In particular, Rh. sp. BCP1 and Rh. opacus R7 were able to utilize NAs such as cyclohexane carboxylic acid and cyclopentane carboxylic acid as the sole carbon and energy sources, even at concentrations up to 1000 mg/L. The presence of either substituents or longer carboxylic acid chains attached to the cyclohexane ring negatively affected the growth by pure bacterial cultures. Moreover, BCP1 and R7 cells incubated in the presence of CHCA or CPCA show a general increase of saturated and methyl-substituted fatty acids in their membrane, while the cis-mono-unsaturated ones decrease, as compared to glucose-grown cells. The observed lipid molecules modification during the growth in the presence of NAs is suggested as a possible mechanism to decrease the fluidity of the cell membrane to counteract NAs toxicity. In order to further evaluate this toxic effect on cell features, the morphological changes of BCP1 and R7 cells were also assessed through Transmission Electron Microscopy (TEM), revealing interesting ultrastructural changes. The induction of putative genes, and the construction of a random transposon mutagenesis library were also carried out to reveal the mechanisms by which these Rhodococcus strains can degrade toxic compounds such as NAs.