La piattaforma alla base di sviluppo e gestione dell'Infrastruttura della Direzione Servizi Informativi

Garantire la continuità di servizio dei sistemi gestionali usati dagli utenti dell’Istituto e favorirne l’evoluzione tecnologica sono due delle principali sfide a cui l'Ufficio Sviluppo e Gestione Strategica dell’Infrastruttura risponde. In questo lavoro viene descritta la piattaforma, creata dall’Ufficio nel corso del tempo, con cui continuità di servizio ed evoluzione tecnologica vengono soddisfatte grazie all’unione fra l’Infrastruttura di Virtualizzazione utilizzata, una serie di Servizi messi a disposizione (es. Monitoraggio e Allarmistica, CICD) e delle Metodologie (es. Efficienza Operativa, Approccio DevOps) che regolano le attività e l’interazione con la piattaforma stessa. Una piattaforma tecnologica, organizzativa e culturale che nel tempo ha fatto emergere un ecosistema in grado di alimentare processi capaci da una parte di operare infrastruttura e servizi in maniera efficiente, dall’altra di promuovere flessibilità ed elasticità nell’evoluzione dei sistemi gestionali informatici usati nell’Istituto

SGSI project at CNAF

The Italian Tier1 center is mainly focused on LHC and physics experiments in general. Recently we tried to widen our area of activity and established a collaboration with the University of Bologna to set-up an area inside our computing center for hosting experiments with high demands of security and privacy requirements on stored data. The first experiment we are going to host is Harmony, a project part of IMI's Big Data for Better Outcomes programme (IMI stands for Innovative Medicines Initiative). In order to be able to accept this kind of data we had to make a subset of our computing center compliant with the ISO 27001 regulation. In this article we will describe the SGSI project (Sistema Gestione Sicurezza Informazioni, Information Security Management System) with details of all the processes we have been through in order to become ISO 27001 compliant, with a particular focus on the separation of the project dedicated resources from all the others hosted in the center. We will also describe the software solutions adopted to allow this project to accept in the future any experiment or collaboration in need for this kind of security procedures

Functional and chemical analysis of the secretory pathway

Protein transport through the secretory pathway is an essential process for all living organisms. While studies over the last three decades have enormously increased our understanding of this event, many of the components involved in the process of protein secretion have yet to be identified, and we are still lacking mechanistic insight into many steps of the secretory pathway. The main objective of this work is to identify new components involved in the secretory pathway. This will be achieved through the establishment of a functional genomics screen in drosophila cells and successive characterization of candidate genes as well as through a pharmacological approach in mammalian cells. Chapter I will describe the functional genomics approach that led to the identification of new components of the secretory pathway. Genome wide RNA interference (RNAi) knockdown was performed on Drosophila cell line stably expressing a secreted form of horseradish peroxidase (HRP). In this cell line, the systematic depletion of the product (mRNA) of genes involved in secretion results in a defect in the release of HRP into the medium. The amount of HRP measured by chemiluminescence provides an indication of the level of secretion upon the loss of a certain gene product. This functional genomics screen approach has led to the identification of approximately 100 genes. The initial characterization of one of these genes is reported. The following chapters will describe the pharmacological approach, which relies on the use of a natural product (norrisolide), a compound able to cause Golgi complex fragmentation and to block secretion in mammalian cells (Brady et al., 2004; Guizzunti et al., 2006; Guizzunti et al., 2007). In chapter II it is introduced norrisolide, a natural product able to induce irreversible fragmentation of the Golgi complex. The Golgi apparatus, a central organelle of the secretory pathway is a dynamic structure whose organization is maintained by a balance of membrane input and output. The fragmentation of the Golgi complex by norrisolide provides a mean to identify the mechanisms by which Golgi organization is maintained and regulated. Norrisolide activity is analyzed through the use of norrisolide-derived probes. We will show how norrisolide's core is necessary and sufficient to induce Golgi fragmentation. A fluorescent analogue of norrisolide will be used to visualize the intracellular localization of norrisolide's target. Chapter III shows the design of trifunctional probes based on norrisolide's structure that can be used to identify norrisolide's target. These probes are designed to contain norrisolide's core, a crosslinking agent and a tag. Different crosslinkers and tags are discusse

Functional and chemical analysis of the secretory pathway

Protein transport through the secretory pathway is an essential process for all living organisms. While studies over the last three decades have enormously increased our understanding of this event, many of the components involved in the process of protein secretion have yet to be identified, and we are still lacking mechanistic insight into many steps of the secretory pathway. The main objective of this work is to identify new components involved in the secretory pathway. This will be achieved through the establishment of a functional genomics screen in drosophila cells and successive characterization of candidate genes as well as through a pharmacological approach in mammalian cells. Chapter I will describe the functional genomics approach that led to the identification of new components of the secretory pathway. Genome wide RNA interference (RNAi) knockdown was performed on Drosophila cell line stably expressing a secreted form of horseradish peroxidase (HRP). In this cell line, the systematic depletion of the product (mRNA) of genes involved in secretion results in a defect in the release of HRP into the medium. The amount of HRP measured by chemiluminescence provides an indication of the level of secretion upon the loss of a certain gene product. This functional genomics screen approach has led to the identification of approximately 100 genes. The initial characterization of one of these genes is reported. The following chapters will describe the pharmacological approach, which relies on the use of a natural product (norrisolide), a compound able to cause Golgi complex fragmentation and to block secretion in mammalian cells (Brady et al., 2004; Guizzunti et al., 2006; Guizzunti et al., 2007). In chapter II it is introduced norrisolide, a natural product able to induce irreversible fragmentation of the Golgi complex. The Golgi apparatus, a central organelle of the secretory pathway is a dynamic structure whose organization is maintained by a balance of membrane input and output. The fragmentation of the Golgi complex by norrisolide provides a mean to identify the mechanisms by which Golgi organization is maintained and regulated. Norrisolide activity is analyzed through the use of norrisolide-derived probes. We will show how norrisolide's core is necessary and sufficient to induce Golgi fragmentation. A fluorescent analogue of norrisolide will be used to visualize the intracellular localization of norrisolide's target. Chapter III shows the design of trifunctional probes based on norrisolide's structure that can be used to identify norrisolide's target. These probes are designed to contain norrisolide's core, a crosslinking agent and a tag. Different crosslinkers and tags are discusse

The fate of PrP GPI-Anchor Signal Peptide is modulated by P238S pathogenic mutation.

: GPI Anchored proteins are localized to the plasma membrane via a C-terminally linked glycosylphosphatidylinositol (GPI) anchor. The GPI anchor is added concomitantly to the cleavage of the carboxy-terminal GPI-anchor signal sequence, thereby causing the release of a C-terminal hydrophobic peptide, whose fate has not yet been investigated. Here we followed the fate of the GPI-attachment signal of the prion protein (PrP), a protein implicated in various types of transmissible neurodegenerative spongiform encephalopathies (TSE). PrP GPI-anchor signal sequence shows a remarkable and unusual degree of conservation across the species and contains two point mutations (M232R/T and P238S) that are responsible for genetic forms of prion disorders. We show that PrP GPI-anchor signal peptide, but not the one from an unrelated GPI-anchored protein (Folate Receptor), undergoes degradation via the proteasome. Moreover, the P238S point mutation partially protects PrP GPI-anchor signal peptide from degradation. Our data provide the first attempt to address the fate of a GPI-anchor signal peptide and identify a role for the P238S mutation, suggesting the possibility that PrP GPI-anchor signal peptide could play a role in neurodegenerative prion diseases

Cytosolically expressed PrP GPI-signal peptide interacts with mitochondria.

International audienceWe previously reported that PrP GPI-anchor signal peptide (GPI-SP) is specifically degraded by the proteasome. Additionally, we showed that the point mutation P238S, responsible for a genetic form of prion diseases, while not affecting the GPI-anchoring process, results in the accumulation of PrP GPI-SP, suggesting the possibility that PrP GPI-anchor signal peptide could play a role in neurodegenerative prion diseases. We now show that PrP GPI-SP, when expressed as a cytosolic peptide, is able to localize to the mitochondria and to induce mitochondrial fragmentation and vacuolarization, followed by loss in mitochondrial membrane potential, ultimately resulting in apoptosis. Our results identify the GPI-SP of PrP as a novel candidate responsible for the impairment in mitochondrial function involved in the synaptic pathology observed in prion diseases, establishing a link between PrP GPI-SP accumulation and neuronal death

Cluvenone induces apoptosis via a direct target in mitochondria: a possible mechanism to circumvent chemo-resistance?

The synthetic caged Garcinia xanthone, cluvenone, has potent and selective cytotoxicity against numerous cancer cell lines including those that are multi-drug resistant. The direct target of this structurally and functionally unique agent is unknown and that of the parent natural product, gambogic acid (GA), presently in clinical trials, is not yet entirely clear. For the first time, using fluorescently labeled GA (GA-Bodipy), we determined that GA-Bodipy localized in mitochondria and was effectively displaced by cluvenone in competition experiments indicating that the direct target of cluvenone resided in mitochondria and was shared by GA. In agreement with these findings, treatment of HeLa cells with cluvenone or GA resulted in disruption of mitochondrial morphology within 4 h. Furthermore, experiments using the potential sensitive JC-1 dye demonstrated that cells treated with 1 μM cluvenone for 1 h had significant loss of MMP compared to control cells. Examination of Cyt c levels in leukemia cells treated with 1 μM cluvenone resulted in a 4-fold increase in levels of both cytosolic and mitochondrial Cyt c. In agreement with Cyt c release, caspase 9 activity was increased 2.6-fold after treatment of cells for 5 h with 1 μM cluvenone. Remarkably, the caspase-9 inhibitor, Z-LEHD-FMK, blocked cluvenone-induced apoptosis in a dose-dependent manner with apoptosis being completely blocked by 10 μM of the inhibitor. In conclusion, cluvenone, an agent with potent cytotoxicity against multi-drug resistant tumor cells, has direct targets in mitochondria thus setting precedence for drug discovery efforts against these targets in the treatment of refractory cancers

Cluvenone induces apoptosis via a direct target in mitochondria: a possible mechanism to circumvent chemo-resistance?

International audienceThe synthetic caged Garcinia xanthone, cluvenone, has potent and selective cytotoxicity against numerous cancer cell lines including those that are multi-drug resistant. The direct target of this structurally and functionally unique agent is unknown and that of the parent natural product, gambogic acid (GA), presently in clinical trials, is not yet entirely clear. For the first time, using fluorescently labeled GA (GA-Bodipy), we determined that GA-Bodipy localized in mitochondria and was effectively displaced by cluvenone in competition experiments indicating that the direct target of cluvenone resided in mitochondria and was shared by GA. In agreement with these findings, treatment of HeLa cells with cluvenone or GA resulted in disruption of mitochondrial morphology within 4 h. Furthermore, experiments using the potential sensitive JC-1 dye demonstrated that cells treated with 1 μM cluvenone for 1 h had significant loss of MMP compared to control cells. Examination of Cyt c levels in leukemia cells treated with 1 μM cluvenone resulted in a 4-fold increase in levels of both cytosolic and mitochondrial Cyt c. In agreement with Cyt c release, caspase 9 activity was increased 2.6-fold after treatment of cells for 5 h with 1 μM cluvenone. Remarkably, the caspase-9 inhibitor, Z-LEHD-FMK, blocked cluvenone-induced apoptosis in a dose-dependent manner with apoptosis being completely blocked by 10 μM of the inhibitor. In conclusion, cluvenone, an agent with potent cytotoxicity against multi-drug resistant tumor cells, has direct targets in mitochondria thus setting precedence for drug discovery efforts against these targets in the treatment of refractory cancers