Shedding light on the role of the prokaryotic assemblage in the biogeochemical cycles of the dark ocean

Se han eliminado las páginas en blanco

The role of the NG2 proteoglycan in migration and characterization of the interaction with the intracellular binding partner, Syntenin

NG2 is a transmembrane proteoglycan with two N-terminal LNS domains and a C-terminal PDZ-binding motif. It is expressed in the developing and adult CNS by oligodendroglial precursor cells and subpopulations of perisynaptic glia and elsewhere by many immature cell types. In order to elucidate the functions of the protein and the heterogenous cell population which expresses it, we undertook to identify and characterise interaction partners of the molecule. The presence of the C-terminal PDZ recognition site in NG2 suggested PDZ-domain proteins as intracellular binding partners. In this work, interaction between the PDZ protein Syntenin and NG2 has been characterised. Syntenin is known to be involved in plasma membrane dynamics, metastasis and adhesion. Syntenin may thus link NG2 to the cytoskeleton, mediating migration of developing oligodendrocytes to axonal tracts prior to myelination, as well as process movement of NG2+ perisynaptic glia. NG2 is involved in cell spreading and polyclonal antibodies against NG2 inhibit the migration of immature glia and cell lines expressing the molecule. In this work we have characterised the segments of the extracellular portion of NG2 that are involved in migration. We found that the extracellular region immediately preceding the transmembrane segment is most important for cell motility. As part of this thesis, biochemical approaches to identify a trans-binding ligand interacting with the extracellular part of NG2 was also explored.

Reverse genetic studies of Benyvirus - Polymyxa betae molecular interaction: Role of the RNA4-encoded protein in virus transmission

Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, is included within viruses transmitted through the soil from plasmodiophorid as Polymyxa betae. BNYVV is the causal agent of Rhizomania, which induces abnormal rootlet proliferation and is widespread in the sugar beet growing areas in Europe, Asia and America; for review see (Peltier et al., 2008). In this latter continent, Beet soil-borne mosaic virus (BSBMV) has been identified (Lee et al., 2001) and belongs to the benyvirus genus together with BNYVV, both vectored by P. betae. BSBMV is widely distributed only in the United States and it has not been reported yet in others countries. It was first identified in Texas as a sugar beet virus morphologically similar but serologically distinct to BNYVV. Subsequent sequence analysis of BSBMV RNAs evidenced similar genomic organization to that of BNYVV but sufficient molecular differences to distinct BSBMV and BNYVV in two different species (Rush et al., 2003). Benyviruses field isolates usually consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs -1 contains a single long ORF encoding polypeptide that shares amino acid homology with known viral RNA-dependent RNA polymerases (RdRp) and helicases. RNAs -2 contains six ORFs: capsid protein (CP), one readthrough protein, triple gene block proteins (TGB) that are required for cell-to-cell virus movement and the sixth 14 kDa ORF is a post-translation gene silencing suppressor. RNAs -3 is involved on disease symptoms and is essential for virus systemic movement. BSBMV RNA-3 can be trans-replicated, trans-encapsidated by the BNYVV helper strain (RNA-1 and -2) (Ratti et al., 2009). BNYVV RNA-4 encoded one 31 kDa protein and is essential for vector interactions and virus transmission by P. betae (Rahim et al., 2007). BNYVV RNA-5 encoded 26 kDa protein that improve virus infections and accumulation in the hosts. We are interest on BSBMV effect on Rhizomania studies using powerful tools as full-length infectious cDNA clones. B-type full-length infectious cDNA clones are available (Quillet et al., 1989) as well as A/P-type RNA-3, -4 and -5 from BNYVV (unpublished). A-type BNYVV full-length clones are also available, but RNA-1 cDNA clone still need to be modified. During the PhD program, we start production of BSBMV full-length cDNA clones and we investigate molecular interactions between plant and Benyviruses exploiting biological, epidemiological and molecular similarities/divergences between BSBMV and BNYVV. During my PhD researchrs we obtained full length infectious cDNA clones of BSBMV RNA-1 and -2 and we demonstrate that they transcripts are replicated and packaged in planta and able to substitute BNYVV RNA-1 or RNA-2 in a chimeric viral progeny (BSBMV RNA-1 + BNYVV RNA-2 or BNYVV RNA-1 + BSBMV RNA-2). During BSBMV full-length cDNA clones production, unexpected 1,730 nts long form of BSBMV RNA-4 has been detected from sugar beet roots grown on BSBMV infected soil. Sequence analysis of the new BSBMV RNA-4 form revealed high identity (~100%) with published version of BSBMV RNA-4 sequence (NC_003508) between nucleotides 1-608 and 1,138-1,730, however the new form shows 528 additionally nucleotides between positions 608-1,138 (FJ424610). Two putative ORFs has been identified, the first one (nucleotides 383 to 1,234), encode a protein with predicted mass of 32 kDa (p32) and the second one (nucleotides 885 to 1,244) express an expected product of 13 kDa (p13). As for BSBMV RNA-3 (Ratti et al., 2009), full-length BSBMV RNA-4 cDNA clone permitted to obtain infectious transcripts that BNYVV viral machinery (Stras12) is able to replicate and to encapsidate in planta. Moreover, we demonstrated that BSBMV RNA-4 can substitute BNYVV RNA-4 for an efficient transmission through the vector P. betae in Beta vulgaris plants, demonstrating a very high correlation between BNYVV and BSBMV. At the same time, using BNYVV helper strain, we studied BSBMV RNA-4’s protein expression in planta. We associated a local necrotic lesions phenotype to the p32 protein expression onto mechanically inoculated C. quinoa. Flag or GFP-tagged sequences of p32 and p13 have been expressed in viral context, using Rep3 replicons, based on BNYVV RNA-3. Western blot analyses of local lesions contents, using FLAG-specific antibody, revealed a high molecular weight protein, which suggest either a strong interaction of BSBMV RNA4’s protein with host protein(s) or post translational modifications. GFP-fusion sequences permitted the subcellular localization of BSBMV RNA4’s proteins. Moreover we demonstrated the absence of self-activation domains on p32 by yeast two hybrid system approaches. We also confirmed that p32 protein is essential for virus transmission by P. betae using BNYVV helper strain and BNYVV RNA-3 and we investigated its role by the use of different deleted forms of p32 protein. Serial mechanical inoculation of wild-type BSBMV on C. quinoa plants were performed every 7 days. Deleted form of BSBMV RNA-4 (1298 bp) appeared after 14 passages and its sequence analysis shows deletion of 433 nucleotides between positions 611 and 1044 of RNA-4 new form. We demonstrated that this deleted form can’t support transmission by P. betae using BNYVV helper strain and BNYVV RNA-3, moreover we confirmed our hypothesis that BSBMV RNA-4 described by Lee et al. (2001) is a deleted form. Interesting after 21 passages we identifed one chimeric form of BSBMV RNA-4 and BSBMV RNA-3 (1146 bp). Two putative ORFs has been identified on its sequence, the first one (nucleotides 383 to 562), encode a protein with predicted mass of 7 kDa (p7), corresponding to the N-terminal of p32 protein encoded by BSBMV RNA-4; the second one (nucleotides 562 to 789) express an expected product of 9 kDa (p9) corresponding to the C-terminal of p29 encoded by BSBMV RNA-3. Results obtained by our research in this topic opened new research lines that our laboratories will develop in a closely future. In particular BSBMV p32 and its mutated forms will be used to identify factors, as host or vector protein(s), involved in the virus transmission through P. betae. The new results could allow selection or production of sugar beet plants able to prevent virus transmission then able to reduce viral inoculum in the soil.

The role of the West African Monsoon in the tropical to mid-latitudes climate

The interaction between atmosphere–land–ocean–biosphere systems plays a prominent role on the atmospheric dynamics and on the convective rainfall distribution over the West Africa monsoon area during the boreal summer. In particular, the initialization of convective systems in the Sub – Sahelian region has been directly linked to soil moisture heterogeneities identified as the major triggering, development and propagation of convective systems. The present study aims at investigating African monsoon large scale convective dynamics and rainfall diurnal cycle through an exploration of the hypothesis behind the mechanisms of a monsoon phenomenon as an emergence of a collective dynamics of many propagating convective systems. Such hypothesis is based on the existence of an internal self – regulation mechanism among the various components. To achieve these results a multiple analysis was performed based on remote sensed rainfall dataset, and global and regional modelling data for a period of 5 seasons: 2004 - 2008. Satellite rainfall data and convective occurrence variability were studied for assessing typical spatio – temporal signatures and characteristics with an emphasis to the diurnal cycle footprint. A global model and regional model simulation datasets, specifically developed for this analysis and based on Regional Atmospheric Modelling System – RAMS, have been analysed. Results from numerical model datasets highlight the evidence of a synchronization between the destabilization of the convective boundary layer and rainfall occurrence due to the solar radiation forcing through the latent heat release. This supports the conclusion that the studied interacting systems are associated with a process of mutual adjustment of rhythms. Furthermore, this rainfall internal coherence was studied in relation to the West African Heat Low pressure system, which has a prominent role in the large scale summer variability over the Mediterranean area since it is acting as one of dynamic link between sub tropical and midlatitudes variability.

Role of the stratospheric dynamics in the southern hemisphere long-term climate change

Il raffreddamento stratosferico associato alla riduzione dell’ozono nelle regioni polari induce un rafforzamento dei venti occidentali nella bassa stratosfera, uno spostamento verso il polo e un’intensificazione del jet troposferico delle medie latitudini. Si riscontra una proiezione di questi cambiamenti a lungo termine sulla polarità ad alto indice di un modo di variabilità climatica, il Southern Annular Mode, alla superficie, dove i venti occidentali alle medie latitudini guidano la Corrente Circumpolare Antartica influenzando la circolazione oceanica meridionale e probabilmente l’estensione del ghiaccio marino ed i flussi di carbonio aria-mare nell’Oceano Meridionale. Una limitata rappresentazione dei processi stratosferici nei modelli climatici per la simulazione del passato e la previsione dei cambiamenti climatici futuri, sembrerebbe portare ad un errore nella rappresentazione dei cambiamenti troposferici a lungo termine nelle rispettive simulazioni. In questa tesi viene condotta un’analisi multi-model mettendo insieme i dati di output derivati da diverse simulazioni di modelli climatici accoppiati oceano-atmosfera, che partecipano al progetto CMIP5, con l'obiettivo di comprendere come le diverse rappresentazioni della dinamica stratosferica possano portare ad una differente rappresentazione dei cambiamenti climatici alla superficie. Vengono utilizzati modelli “High Top” (HT), che hanno una buona rappresentazione della dinamica stratosferica, e modelli “Low Top” (LT), che invece non ne hanno. I risultati vengono confrontati con le reanalisi meteorologiche globali disponibili (ERA-40). Viene mostrato come la rappresentazione e l’intensità del raffreddamento radiativo iniziale e di quello dinamico nella bassa stratosfera, nei modelli, siano i fattori chiave che controllano la successiva risposta troposferica, e come il raffreddamento stesso dipenda dalla rappresentazione della dinamica stratosferica. Si cerca inoltre di differenziare i modelli in base alla loro rappresentazione del raffreddamento radiativo e dinamico nella bassa stratosfera e alla risposta del jet troposferico. Nei modelli, si riscontra che il trend del jet nell'intera troposfera è significativamente correlato linearmente al raffreddamento stesso della bassa stratosfera.

The role of the NadR regulator during infection and its implication for the coverage of a new Meningococcus B vaccine

Background: Neisseria meningitides represents a major cause of meningitis and sepsis. The meningococcal regulator NadR was previously shown to repress the expression of the Neisserial Adhesin A (NadA) and play a major role in its phase-variation. NadA is a surface exposed protein involved in epithelial cell adhesion and colonization and a major component of 4CMenB, a novel vaccine to prevent meningococcus serogroup B infection. The NadR mediated repression of NadA is attenuated by 4-HPA, a natural molecule released in human saliva. Results: In this thesis we investigated the global role of NadR during meningogoccal infection, identifying through microarray analysis the NadR regulon. Two distinct types of NadR targets were identified, differing in their promoter architectures and 4HPA responsive activities: type I are induced, while type II are co-repressed in response to the same 4HPA signal. We then investigate the mechanism of regulation of NadR by 4-HPA, generating NadR mutants and identifying classes or residues involved in either NadR DNA binding or 4HPA responsive activities. Finally, we studied the impact of NadR mediated repression of NadA on the vaccine coverage of 4CMenB. A selected MenB strains is not killed by sera from immunized infants when the strain is grown in vitro, however, in an in vivo passive protection model, the same sera protected infant rats from bacteremia. Finally, using bioluminescent reporters, nadA expression in the infant rat model was induced in vivo at 3 h post-infection. Conclusions: Our results suggest that NadR coordinates a broad transcriptional response to signals present in the human host, enabling the meningococcus to adapt to the relevant host niche. During infectious disease the effect of the same signal on NadR changes between different targets. In particular NadA expression is induced in vivo, leading to efficient killing of meningococcus by anti-NadA antibodies elicited by the 4CMenB vaccine.

Role of the Transcription Factor Sox2 in the Osteogenic Lineage

The Sox2 transcription factor is modified by sumoylation at the K247 position although the addition of SUMO1 and Pias1 promotes the sumoylation of Sox2 at the additional K123 site. The role of sumoylation on Sox2 biological functions was analyzed by comparing the activity of WT and sumoylation mutants on the transcription of the FGF4 gene in HeLa cells and on the downregulation of the Wnt pathwayvin 293T cells. When SUMO1 and PIAS1 promote the sumoylation of WT Sox2, the transcriptional activity of the FGF4 promoter is inhibited showing that Sox2 sumoylation is necessary for the repression function. However, there is no effect of Sox2 sumoylation on β-Catenin activity. Since we were interested in osteoblast differentiation we set up an inducible system for Sox2 in primary osteoblasts. Following Sox2 doxycycline induction, 158 genes were differentially expressed: 120 up-regulated and 38 down-regulated. We annotated as direct Sox2 targets a number of genes involved in osteoblast biology and we further analyzed 3 of them involved in the BMP pathway. The results show that Sox2 regulates the BMP pathway without affecting SMAD phosphorylation, and that Sox2 sumoylation is not necessary for this function. We also found that genes involved in the Hippo pathway were direct Sox2 targets. As the Hippo pathway is activated by Sox2 and Sox2 interacts with the NF2 promoter, we checked the effect of Sox2 on the expression of NF2. We showed that Sox2 down-regulates the transcriptional activity of the NF2 promoter, allowing the transcription of the YAP/TEAD genes in osteoblasts, thus acting as an upstream regulator of the Hippo pathway. We conclude that Sox2 induction in osteoblasts triggers FGF dependent inhibition of the BMP, Wnt and Hippo pathways.

Environmental impact assessment of the School of Engineering and Architecture (Lazzaretto) - A pathway towards LEED certification

The current work has for object the improvement and the maintenance of the School of Engineering and Architecture in Via Terracini 28 (Bologna), with the prospective to maximize the operative efficiency reducing to the minimum the environmental impact and the costs. In order to realize this work the LEED certification has been used. LEED (Leadership in Energy and Environmental Design) is a certification system of the buildings. It was born in United States by the U.S. Green Building Council (USGBC)

Control of Steroid 21-oic Acid Synthesis by Peroxisome Proliferator-activated Receptor and Role of the Hypothalamic-Pituitary-Adrenal Axis

A previous study identified the peroxisome proliferator-activated receptor alpha (PPARalpha) activation biomarkers 21-steroid carboxylic acids 11beta-hydroxy-3,20-dioxopregn-4-en-21-oic acid (HDOPA) and 11beta,20-dihydroxy-3-oxo-pregn-4-en-21-oic acid (DHOPA). In the present study, the molecular mechanism and the metabolic pathway of their production were determined. The PPARalpha-specific time-dependent increases in HDOPA and 20alpha-DHOPA paralleled the development of adrenal cortex hyperplasia, hypercortisolism, and spleen atrophy, which was attenuated in adrenalectomized mice. Wy-14,643 activation of PPARalpha induced hepatic FGF21, which caused increased neuropeptide Y and agouti-related protein mRNAs in the hypothalamus, stimulation of the agouti-related protein/neuropeptide Y neurons, and activation of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in increased adrenal cortex hyperplasia and corticosterone production, revealing a link between PPARalpha and the HPA axis in controlling energy homeostasis and immune regulation. Corticosterone was demonstrated as the precursor of 21-carboxylic acids both in vivo and in vitro. Under PPARalpha activation, the classic reductive metabolic pathway of corticosterone was suppressed, whereas an alternative oxidative pathway was uncovered that leads to the sequential oxidation on carbon 21 resulting in HDOPA. The latter was then reduced to the end product 20alpha-DHOPA. Hepatic cytochromes P450, aldehyde dehydrogenase (ALDH3A2), and 21-hydroxysteroid dehydrogenase (AKR1C18) were found to be involved in this pathway. Activation of PPARalpha resulted in the induction of Aldh3a2 and Akr1c18, both of which were confirmed as target genes through introduction of promoter luciferase reporter constructs into mouse livers in vivo. This study underscores the power of mass spectrometry-based metabolomics combined with genomic and physiologic analyses in identifying downstream metabolic biomarkers and the corresponding upstream molecular mechanisms.

The role of the interplay between polymer architecture and bacterial surface properties on the microbial adhesion to polyoxazoline-based ultrathin films

Surface platforms were engineered from poly(L-lysine)-graft-poly(2-methyl-2-oxazoline) (PLL-g-PMOXA) copolymers to study the mechanisms involved in the non-specific adhesion of Escherichia coli (E. coli) bacteria. Copolymers with three different grafting densities (PMOXA chains/Lysine residue of 0.09, 0.33 and 0.56) were synthesized and assembled on niobia (Nb O ) surfaces. PLL-modified and bare niobia surfaces served as controls. To evaluate the impact of fimbriae expression on the bacterial adhesion, the surfaces were exposed to genetically engineered E. coli strains either lacking, or constitutively expressing type 1 fimbriae. The bacterial adhesion was strongly influenced by the presence of bacterial fimbriae. Non-fimbriated bacteria behaved like hard, charged particles whose adhesion was dependent on surface charge and ionic strength of the media. In contrast, bacteria expressing type 1 fimbriae adhered to the substrates independent of surface charge and ionic strength, and adhesion was mediated by non-specific van der Waals and hydrophobic interactions of the proteins at the fimbrial tip. Adsorbed polymer mass, average surface density of the PMOXA chains, and thickness of the copolymer films were quantified by optical waveguide lightmode spectroscopy (OWLS) and variable-angle spectroscopic ellipsometry (VASE), whereas the lateral homogeneity was probed by time-of-flight secondary ion mass spectrometry (ToF-SIMS). Streaming current measurements provided information on the charge formation of the polymer-coated and the bare niobia surfaces. The adhesion of both bacterial strains could be efficiently inhibited by the copolymer film only with a grafting density of 0.33 characterized by the highest PMOXA chain surface density and a surface potential close to zero.