Intracellular distribution of Tankyrases as detected by multicolor immunofluorescence techniques

Poly(ADP-ribose) polymerases are a family of enzymes that catalyze the conversion of NAD+ into ADP-ribose. Among them, Tankyrases have been found to bind to centrosome, mitotic spindle and microsome proteins, in the cytoplasm, and to telomeres in the nucleus, where they play a relevant role in telomere metabolism. However, their precise intracellular localization during interphase has not been so far fully elucidated. We investigated this aspect in situ by double immunofluorescence experiments using antibodies recognizing Tankyrases 1–2 or other proteins residing in specific organelles (Golgi apparatus, mitochondria, lysosomes, endoplasmic reticulum). We used HeLa cells as a model system in vitro, before and after treatment with either actinomycin D or etoposide, to also investigate the possible relocation of Tankyrases during apoptosis. We observed that Tankyrases are distributed both in the nucleus and in the cytoplasm; in this latter compartment, they were found to colocate with the Golgi apparatus but never with the mitochondria; a pool of Tankyrases also colocates with the endoplasmic reticulum and lysosomes. Interestingly, in cells with clear signs of apoptosis, Tankyrases were detectable in the cytoplasmic blebs: this suggests that they are not massively cleaved during apoptosis and persist in the largely heterogeneous apoptotic remnants which are known to contain components of cytoplasmic and nuclear origin

The Golgi apparatus is a primary site of intracellular damage after photosensitization with Rose Bengal acetate

The aim of the present investigation was to elucidate whether the Golgi apparatus undergoes photodamage following administration of the fluorogenic substrates Rose Bengal acetate (RBAc) and irradiation at the appropriate wavelength. Human HeLa cells were treated in culture and the changes in the organization of the Golgi apparatus were studied using fluorescence confocal microscopy and electron microscopy, after immunocytochemical labeling. To see whether the cytoskeletal components primarily involved in vescicle traffic (i.e., microtubules) might also be affected, experiments of tubulin immunolabeling were performed. After treatment with RBAc and irradiation, cells were allowed to grow in drug-free medium for different times. 24hr after irradiation, the cisternae of the Golgi apparatus became packed, and after 48-72 hr they appeared more fragmented and scattered throughout the cytoplasm; these changes in the organization of the Golgi cisternae were confirmed at electron microscopy. Interestingly enough, apoptosis was found to occur especially 48-72h after irradiation, and apoptotic cells exhibited a dramatic fragmentation of the Golgi membranes. The immunolabeling with anti-tubulin antibody showed that microtubules were also affected by irradiation in RBAc-treated cells

Nuclear localization of phosphorylated c-Myc protein in human tumor cells.

Using immunocytochemical techniques at light and electron microscopy, we analysed the distribution of phosphorylated c-Myc in actively proliferating human HeLa cells. The distribution pattern of c-Myc was also compared with those of other ribonucleoprotein (RNP)-containing components (PANA, hnRNP-core proteins, fibrillarin) or RNP-associated nuclear proteins (SC-35 splicing factor). Our results provide the first evidence that phosphorylated c-Myc accumulates in the nucleus of tumor cells, where it colocalizes with fibrillarin, both in the nucleolus and in extranucleolar structures

SplitBox: Toward Efficient Private Network Function Virtualization

This paper presents SplitBox, an efficient system for privacy-preserving processing of network functions that are outsourced as software processes to the cloud. Specifically, cloud providers processing the network functions do not learn the network policies instructing how the functions are to be processed. First, we propose an abstract model of a generic network function based on match-action pairs. We assume that this function is processed in a distributed manner by multiple honest-but-curious cloud service providers. Then, we introduce our SplitBox system for private network function virtualization and present a proof-of-concept implementation on FastClick, an extension of the Click modular router, using a firewall as a use case. Our experimental results achieve a throughput of over 2 Gbps with 1 kB-sized packets on average, traversing up to 60 firewall rules

Multicolor fluorescence technique to detect apoptotic cells in advanced coronary atherosclerotic plaques.

Apoptosis occurring in atherosclerotic lesions has been suggested to be involved in the evolution and the structural stability of the plaques. It is still a matter of debate whether apoptosis mainly involves vascular smooth muscle cells (vSMCs) in the fibrous tissue or inflammatory (namely foam) cells, thus preferentially affecting the cell-poor lipid core of the atherosclerotic plaques. The aim of the present investigation was to detect the presence of apoptotic cells and to estimate their percentage in a series of atherosclerotic plaques obtained either by autopsy or during surgical atherectomy. Apoptotic cells were identified on paraffinembedded sections on the basis of cell nuclear morphology after DNA staining and/or by cytochemical reactions (TUNEL assay, immunodetection of the proteolytic poly (ADP-ribose) polymerase-1 [PARP-1] fragment); biochemical procedures (identifying DNA fragmentation or PARP-1 proteolysis) were also used. Indirect immunofluorescence techniques were performed to label specific antigens for either vSMCs or macrophages (i.e., the cells which are most likely prone to apoptosis in atherosclerotic lesions): the proper selection of fluorochrome labeling allowed the simultaneous detection of the cell phenotype and the apoptotic characteristics, by multicolor fluorescence techniques. Apoptotic cells proved to be less than 5% of the whole cell population, in atherosclerotic plaque sections: this is, in fact, a too low cell fraction to be detected by widely used biochemical methods, such as agarose gel electrophoresis of low-molecular-weight DNA or Western-blot analysis of PARP-1 degradation. Most apoptotic cells were of macrophage origin, and clustered in the tunica media, near or within the lipid-rich core; only a few TUNEL-positive cells were labeled for antigens specific for vSMCs. These results confirm that, among the cell populations in atherosclerotic plaques, macrophage foam-cells are preferentially involved in apoptosis. Their death may decrease the cell number in the lipid core and generate a possibly defective apoptotic clearance: the resulting release of matrix-degrading enzymes could contribute to weakening the fibrous cap and promote the plaque rupture with the risk of acute ischemic events, while increasing the thrombogenic pultaceous pool of the plaque core

Misure ambientali in mare aperto: sviluppo di tecnologie per l'acquisizione e l'analisi di dati meteo-mareografici misurati da una boa oceanografica in Mar Ligure

Obiettivo del presente lavoro è lo sviluppo di un metodo di analisi in grado di stimare i parametri fondamentali del moto ondoso, a partire dai dati acquisiti da tre altimetri acustici installati a bordo di una boa meteo-oceanografica operante in mare aperto. Il metodo di analisi sviluppato compie opportune operazioni di filtraggio sulle sequenze temporali delle misure effettuate dai tre altimetri, quindi, elaborando tali sequenze, fornisce le stime di alcuni parametri caratteristici del moto ondoso (tra cui l’altezza e la direzione di propagazione). I diversi tipi di filtraggio ed il metodo di stima sviluppati si basano sul calcolo di alcuni parametri statistici (tra cui media, mediana e deviazione standard) delle serie temporali di dati acquisiti, sulla conoscenza delle loro densità spettrali di potenza (calcolate mediante FFT), e sul calcolo delle funzioni di crosscorrelazione delle sequenze di dati prese a due a due. Il procedimento di stima realizzato è stato sperimentato su una notevole quantità di dati reali acquisiti in Mar Ligure tramite l’utilizzo della stazione di misura fissa su cui sono montati gli altimetri acustici, ed ha fornito risultati soddisfacenti per quanto riguarda affidabilità e precisione. Nell’ambito della valutazione delle prestazioni del sistema di acquisizione dati e del metodo di stima, i risultati ottenuti sono stati confrontati con misure provenienti da altri sensori a bordo della stazione e con stime analoghe effettuate a partire dai dati acquisiti da un’altra stazione di misura, operante anch’essa in Mar Ligure, ma dotata di strumentazione di altro tipo

Component-aware Orchestration of Cloud-based Enterprise Applications, from TOSCA to Docker and Kubernetes

Enterprise IT is currently facing the challenge of coordinating the management of complex, multi-component applications across heterogeneous cloud platforms. Containers and container orchestrators provide a valuable solution to deploy multi-component applications over cloud platforms, by coupling the lifecycle of each application component to that of its hosting container. We hereby propose a solution for going beyond such a coupling, based on the OASIS standard TOSCA and on Docker. We indeed propose a novel approach for deploying multi-component applications on top of existing container orchestrators, which allows to manage each component independently from the container used to run it. We also present prototype tools implementing our approach, and we show how we effectively exploited them to carry out a concrete case study

Two-color fluorescence detection of Poly (ADP-Ribose) Polymerase-1 (PARP-1) cleavage and DNA strand breaks in etoposide-induced apoptotic cells

During apoptosis, the nuclear enzyme Poly(ADPRibose) Polymerase-1 (PARP-1) catalyzes the rapid and transient synthesis of poly(ADP-ribose) from NAD+ and becomes inactive when cleaved by caspases. The regulation of these two opposite roles of PARP-1 is still unknown. We have recently investigated PARP-1 activation/degradation in Hep-2 cells driven to apoptosis by actinomycin D. In the present work, we have extended our analysis to the effect of the DNA damaging agent etoposide, and paid attention to the relationship between PARP-1 cleavage and DNA fragmentation. An original fluorescent procedure was developed to simultaneously identify in situ the p89 proteolytic fragment of PARP-1 (by immunolabeling) and DNA degradation (by the TUNEL assay). The presence of p89 was observed both in cells with advanced signs of apoptosis (where the PARP-1 fragment is extruded from the nucleus into the cytoplasm) and in TUNEL-negative cells, with only incipient signs of chromatin condensation; this evidence indicates that PARP-1 degradation in etoposide-treated apoptotic cells may precede DNA cleavage