Profiling Three-Dimensional Nuclear Telomeric Architecture of Myelodysplastic Syndromes and Acute Myeloid Leukemia Defines Patient Subgroups

Purpose: Myelodysplastic syndromes (MDS) are a group of disorders characterized by cytopenias, with a propensity for evolution into acute myeloid leukemias (AML). This transformation is driven by genomic instability, but mechanisms remain unknown. Telomere dysfunction might generate genomic instability leading to cytopenias and disease progression. Experimental Design: We undertook a pilot study of 94 patients with MDS (56 patients) and AML (38 patients). The MDS cohort consisted of refractory cytopenia with multilineage dysplasia (32 cases), refractory anemia (12 cases), refractory anemia with excess of blasts (RAEB) 1 (8 cases), RAEB2 (1 case), refractory anemia with ring sideroblasts (2 cases), and MDS with isolated del(5q) (1 case). The AML cohort was composed of AML-M4 (12 cases), AML-M2 (10 cases), AML-M5 (5 cases), AML-M0 (5 cases), AML-M1 (2 cases), AML-M4eo (1 case), and AML with multidysplasia-related changes (1 case). Three-dimensional quantitative FISH of telomeres was carried out on nuclei from bone marrow samples and analyzed using TeloView. Results: We defined three-dimensional nuclear telomeric profiles on the basis of telomere numbers, telomeric aggregates, telomere signal intensities, nuclear volumes, and nuclear telomere distribution. Using these parameters, we blindly subdivided the MDS patients into nine subgroups and the AML patients into six subgroups. Each of the parameters showed significant differences between MDS and AML. Combining all parameters revealed significant differences between all subgroups. Three-dimensional telomeric profiles are linked to the evolution of telomere dysfunction, defining a model of progression from MDS to AML. Conclusions: Our results show distinct three-dimensional telomeric profiles specific to patients with MDS and AML that help subgroup patients based on the severity of telomere dysfunction highlighted in the profiles. Clin Cancer Res; 18(12); 3293-304. (C) 2012 AACR.

Signal transduction in Plasmodium-Red Blood Cells interactions and in cytoadherence

Malaria is responsible for more than 1.5 million deaths each year, especially among children (Snow et al. 2005). Despite of the severity of malaria situation and great effort to the development of new drug targets (Yuan et al. 2011) there is still a relative low investment toward antimalarial drugs. Briefly there are targets classes of antimalarial drugs currently being tested including: kinases, proteases, ion channel of GPCR, nuclear receptor, among others (Gamo et al. 2010). Here we review malaria signal transduction pathways in Red Blood Cells (RBC) as well as infected RBCs and endothelial cells interactions, namely cytoadherence. The last process is thought to play an important role in the pathogenesis of severe malaria. The molecules displayed on the surface of both infected erythrocytes (IE) and vascular endothelial cells (EC) exert themselves as important mediators in cytoadherence, in that they not only induce structural and metabolic changes on both sides, but also trigger multiple signal transduction processes, leading to alteration of gene expression, with the balance between positive and negative regulation determining endothelial pathology during a malaria infection.

Sviluppo di metodologie di localizzazione nel contesto del Comprehensive Nuclear Test-Ban Treaty

Since the first underground nuclear explosion, carried out in 1958, the analysis of seismic signals generated by these sources has allowed seismologists to refine the travel times of seismic waves through the Earth and to verify the accuracy of the location algorithms (the ground truth for these sources was often known). Long international negotiates have been devoted to limit the proliferation and testing of nuclear weapons. In particular the Treaty for the comprehensive nuclear test ban (CTBT), was opened to signatures in 1996, though, even if it has been signed by 178 States, has not yet entered into force, The Treaty underlines the fundamental role of the seismological observations to verify its compliance, by detecting and locating seismic events, and identifying the nature of their sources. A precise definition of the hypocentral parameters represents the first step to discriminate whether a given seismic event is natural or not. In case that a specific event is retained suspicious by the majority of the State Parties, the Treaty contains provisions for conducting an on-site inspection (OSI) in the area surrounding the epicenter of the event, located through the International Monitoring System (IMS) of the CTBT Organization. An OSI is supposed to include the use of passive seismic techniques in the area of the suspected clandestine underground nuclear test. In fact, high quality seismological systems are thought to be capable to detect and locate very weak aftershocks triggered by underground nuclear explosions in the first days or weeks following the test. This PhD thesis deals with the development of two different seismic location techniques: the first one, known as the double difference joint hypocenter determination (DDJHD) technique, is aimed at locating closely spaced events at a global scale. The locations obtained by this method are characterized by a high relative accuracy, although the absolute location of the whole cluster remains uncertain. We eliminate this problem introducing a priori information: the known location of a selected event. The second technique concerns the reliable estimates of back azimuth and apparent velocity of seismic waves from local events of very low magnitude recorded by a trypartite array at a very local scale. For the two above-mentioned techniques, we have used the crosscorrelation technique among digital waveforms in order to minimize the errors linked with incorrect phase picking. The cross-correlation method relies on the similarity between waveforms of a pair of events at the same station, at the global scale, and on the similarity between waveforms of the same event at two different sensors of the try-partite array, at the local scale. After preliminary tests on the reliability of our location techniques based on simulations, we have applied both methodologies to real seismic events. The DDJHD technique has been applied to a seismic sequence occurred in the Turkey-Iran border region, using the data recorded by the IMS. At the beginning, the algorithm was applied to the differences among the original arrival times of the P phases, so the cross-correlation was not used. We have obtained that the relevant geometrical spreading, noticeable in the standard locations (namely the locations produced by the analysts of the International Data Center (IDC) of the CTBT Organization, assumed as our reference), has been considerably reduced by the application of our technique. This is what we expected, since the methodology has been applied to a sequence of events for which we can suppose a real closeness among the hypocenters, belonging to the same seismic structure. Our results point out the main advantage of this methodology: the systematic errors affecting the arrival times have been removed or at least reduced. The introduction of the cross-correlation has not brought evident improvements to our results: the two sets of locations (without and with the application of the cross-correlation technique) are very similar to each other. This can be commented saying that the use of the crosscorrelation has not substantially improved the precision of the manual pickings. Probably the pickings reported by the IDC are good enough to make the random picking error less important than the systematic error on travel times. As a further justification for the scarce quality of the results given by the cross-correlation, it should be remarked that the events included in our data set don’t have generally a good signal to noise ratio (SNR): the selected sequence is composed of weak events ( magnitude 4 or smaller) and the signals are strongly attenuated because of the large distance between the stations and the hypocentral area. In the local scale, in addition to the cross-correlation, we have performed a signal interpolation in order to improve the time resolution. The algorithm so developed has been applied to the data collected during an experiment carried out in Israel between 1998 and 1999. The results pointed out the following relevant conclusions: a) it is necessary to correlate waveform segments corresponding to the same seismic phases; b) it is not essential to select the exact first arrivals; and c) relevant information can be also obtained from the maximum amplitude wavelet of the waveforms (particularly in bad SNR conditions). Another remarkable point of our procedure is that its application doesn’t demand a long time to process the data, and therefore the user can immediately check the results. During a field survey, such feature will make possible a quasi real-time check allowing the immediate optimization of the array geometry, if so suggested by the results at an early stage.

Topological aspects of the human protein interaction network

It is currently widely accepted that the understanding of complex cell functions depends on an integrated network theoretical approach and not on an isolated view of the different molecular agents. Aim of this thesis was the examination of topological properties that mirror known biological aspects by depicting the human protein network with methods from graph- and network theory. The presented network is a partial human interactome of 9222 proteins and 36324 interactions, consisting of single interactions reliably extracted from peer-reviewed scientific publications. In general, one can focus on intra- or intermodular characteristics, where a functional module is defined as "a discrete entity whose function is separable from those of other modules". It is found that the presented human network is also scale-free and hierarchically organised, as shown for yeast networks before. The interactome also exhibits proteins with high betweenness and low connectivity which are biologically analyzed and interpreted here as shuttling proteins between organelles (e.g. ER to Golgi, internal ER protein translocation, peroxisomal import, nuclear pores import/export) for the first time. As an optimisation for finding proteins that connect modules, a new method is developed here based on proteins located between highly clustered regions, rather than regarding highly connected regions. As a proof of principle, the Mediator complex is found in first place, the prime example for a connector complex. Focusing on intramodular aspects, the measurement of k-clique communities discriminates overlapping modules very well. Twenty of the largest identified modules are analysed in detail and annotated to known biological structures (e.g. proteasome, the NFκB-, TGF-β complex). Additionally, two large and highly interconnected modules for signal transducer and transcription factor proteins are revealed, separated by known shuttling proteins. These proteins yield also the highest number of redundant shortcuts (by calculating the skeleton), exhibit the highest numbers of interactions and might constitute highly interconnected but spatially separated rich-clubs either for signal transduction or for transcription factors. This design principle allows manifold regulatory events for signal transduction and enables a high diversity of transcription events in the nucleus by a limited set of proteins. Altogether, biological aspects are mirrored by pure topological features, leading to a new view and to new methods that assist the annotation of proteins to biological functions, structures and subcellular localisations. As the human protein network is one of the most complex networks at all, these results will be fruitful for other fields of network theory and will help understanding complex network functions in general.

Valorisation of organic waste: new developments from proton nuclear magnetic resonance characterization

The last half-century has seen a continuing population and consumption growth, increasing the competition for land, water and energy. The solution can be found in the new sustainability theories, such as the industrial symbiosis and the zero waste objective. Reducing, reusing and recycling are challenges that the whole world have to consider. This is especially important for organic waste, whose reusing gives interesting results in terms of energy release. Before reusing, organic waste needs a deeper characterization. The non-destructive and non-invasive features of both Nuclear Magnetic Resonance (NMR) relaxometry and imaging (MRI) make them optimal candidates to reach such characterization. In this research, NMR techniques demonstrated to be innovative technologies, but an important work on the hardware and software of the NMR LAGIRN laboratory was initially done, creating new experimental procedures to analyse organic waste samples. The first results came from soil-organic matter interactions. Remediated soils properties were described in function of the organic carbon content, proving the importance of limiting the addition of further organic matter to not inhibit soil processes as nutrients transport. Moreover NMR relaxation times and the signal amplitude of a compost sample, over time, showed that the organic matter degradation of compost is a complex process that involves a number of degradation kinetics, as a function of the mix of waste. Local degradation processes were studied with enhanced quantitative relaxation technique that combines NMR and MRI. The development of this research has finally led to the study of waste before it becomes waste. Since a lot of food is lost when it is still edible, new NMR experiments studied the efficiency of conservation and valorisation processes: apple dehydration, meat preservation and bio-oils production. All these results proved the readiness of NMR for quality controls on a huge kind of organic residues and waste.

Contamination of Alpine snow and ice at Colle Gnifetti, Swiss/Italian Alps, from nuclear weapons tests

Plutonium is present in the environment as a consequence of atmospheric nuclear tests, nuclear weapons production and industrial releases over the past 50 years. To study temporal trends, a high resolution Pu record was obtained by analyzing 52 discrete samples of an alpine firn/ice core from Colle Gnifetti (Monte Rosa, 4450 m a.s.l.), dating from 1945 to 1990. The 239Pu signal was recorded directly, without decontamination or preconcentration steps, using an Inductively Coupled Plasma - Sector Field Mass Spectrometer (ICP-SFMS) equipped with an high efficiency sample introduction system, thus requiring much less sample preparation than previously reported methods. The 239Pu profile reflects the three main periods of atmospheric nuclear weapons testing: the earliest peak lasted from 1954/55 to 1958 and was caused by the first testing period reaching a maximum in 1958. Despite a temporary halt of testing in 1959/60, the Pu concentration decreased only by half with respect to the 1958 peak due to long atmospheric residence times. In 1961/62 Pu concentrations rapidly increased reaching a maximum in 1963, which was about 40% more intense than the 1958 peak. After the signing of the "Limited Test Ban Treaty" between USA and USSR in 1964, Pu deposition decreased very sharply reaching a minimum in 1967. The third period (1967-1975) is characterized by irregular Pu concentrations with smaller peaks (about 20-30% of the 1964 peak) which might be related to the deposition of Saharan dust contaminated by the French nuclear tests of the 1960s. The data presented are in very good agreement with Pu profiles previously obtained from the Col du Dome ice core (by multi-collector ICP-MS) and Belukha ice core (by Accelerator Mass Spectrometry, AMS). Although a semi-quantitative method was employed here, the results are quantitatively comparable to previously published results.

The nuclear factor-kappaB and p53 pathways function independently in primary cells and transformed fibroblasts responding to genotoxic damage

With nuclear factor-kappaB (NF-kappaB) and p53 functions generally having disparate outcomes for cell survival and cell division, understanding how these pathways are coordinated following a common activation signal such as DNA damage has important implications for cancer therapy. Conflicting reports concerning NF-kappaB and p53 interplay in different cell line models prompted a reexamination of this issue using mouse primary thymocytes and embryonic fibroblasts, plus fibroblasts transformed by E1A12S. Here, we report that following the treatment of these cells with a range of stress stimuli, p53 and NF-kappaB were found to regulate cell cycling and survival independently.

Promoter- and RNA polymerase II-dependent hsp-16 gene association with nuclear pores in Caenorhabditis elegans.

Some inducible yeast genes relocate to nuclear pores upon activation, but the general relevance of this phenomenon has remained largely unexplored. Here we show that the bidirectional hsp-16.2/41 promoter interacts with the nuclear pore complex upon activation by heat shock in the nematode Caenorhabditis elegans. Direct pore association was confirmed by both super-resolution microscopy and chromatin immunoprecipitation. The hsp-16.2 promoter was sufficient to mediate perinuclear positioning under basal level conditions of expression, both in integrated transgenes carrying from 1 to 74 copies of the promoter and in a single-copy genomic insertion. Perinuclear localization of the uninduced gene depended on promoter elements essential for induction and required the heat-shock transcription factor HSF-1, RNA polymerase II, and ENY-2, a factor that binds both SAGA and the THO/TREX mRNA export complex. After induction, colocalization with nuclear pores increased significantly at the promoter and along the coding sequence, dependent on the same promoter-associated factors, including active RNA polymerase II, and correlated with nascent transcripts.

Response of the XENON100 Dark Matter Detector to Nuclear Recoils

Results from the nuclear recoil calibration of the XENON100 dark matter detector installed underground at the Laboratori Nazionali del Gran Sasso, Italy are presented. Data from measurements with an external AmB(241)e neutron source are compared with a detailed Monte Carlo simulation which is used to extract the energy-dependent charge-yield Q(y) and relative scintillation efficiency L-eff. A very good level of absolute spectral matching is achieved in both observable signal channels-scintillation S1 and ionization S2-along with agreement in the two-dimensional particle discrimination space. The results confirm the validity of the derived signal acceptance in earlier reported dark matter searches of the XENON100 experiment.

Stimulation of monocytes by placental microparticles involves toll-like receptors and nuclear factor kappa-light-chain-enhancer of activated B cells.

Human pregnancy is accompanied by a mild systemic inflammatory response, which includes the activation of monocytes circulating in maternal blood. This response is exaggerated in preeclampsia, a placental-dependent disorder specific to human pregnancies. We and others showed that placental syncytiotrophoblast membrane microparticles (STBM) generated in vitro from normal placentas stimulated peripheral blood monocytes, which suggest a contribution of STBM to the systemic maternal inflammation. Here, we analyzed the inflammatory potential of STBM prepared from preeclamptic placentas on primary monocytes and investigated the mode of action in vitro. STBM generated in vitro by placental villous explants of normal or preeclamptic placentas were co-incubated with human peripheral blood monocytes. In some cases, inhibitors of specific cellular functions or signaling pathways were used. The analysis of the monocytic response was performed by flow cytometry, enzyme-linked immunoassays, real-time PCR, and fluorescence microscopy. STBM derived from preeclamptic placentas up-regulated the cell surface expression of CD54, and stimulated the secretion of the pro-inflammatory interleukin (IL)-6 and IL-8 in a similar, dose-dependent manner as did STBM prepared from normal placentas. STBM bound to the cell surface of monocytes, but phagocytosis was not necessary for activation. STBM-induced cytokine secretion was impaired in the presence of inhibitors of toll-like receptor (TLR) signaling or when nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation was blocked. Our results suggest that the inflammatory reaction in monocytes may be initiated by the interaction of STBM with TLRs, which in turn signal through NF-κB to mediate the transcription of genes coding for pro-inflammatory factors.

Transcriptional upregulation of the p21Cip1 promoter by STAT3 and nuclear ErbB2

Over-expression of the receptor tyrosine kinase ErbB2 is prevalent in approximately 30% of human breast carcinomas and confers Taxol resistance. In breast cancer cells, Taxol induces tubulin polymerization and hyperstable microtubule formation. This in turn prematurely activates Cdc2 kinase allowing early entry into the G2/M phase of the cell cycle resultant in mitotic catastrophe followed by apoptosis. Over-expression of ErbB2 upregulates p21Cip1, which inhibits Cdc2 activation, and leads to Taxol resistance in patients. However, the mechanism of ErbB2-mediated p21 Cip1 upregulation is unclear. Here in this study, we investigated the mechanism of ErbB2 downstream signaling events leading to upregulation. The CDKN1A (p21Cip1) gene promoter contains numerous cis-elements including a Signal transducer and activator of transcription (STAT) Inducable Element (SIE) located at -679 kb. Our studies showed ErbB2 overexpressing cells had increased activated levels of STAT3, and therefore we hypothesized that STAT3 is responsible for the upregulation of the p21Cip1 promoter by ErbB2. EMSA and ChIP assays confirmed the binding of STAT3 to the p21Cip1 promoter and luciferase assays showed higher p21 Cip1 promoter activity in ErbB2 over-expressing transfectants when compared to parental cells, in a STAT3 binding site dependant manner. Additionally, reduced level of STAT3 led to reduced p21Cip1 protein expression and promoter activity indicating that both the STAT3 binding site and STAT3 protein are required for ErbB2-mediated p21Cip1 upregulation. Further investigation of ErbB2 downstream signaling showed increased Src kinase activity in ErbB2 over-expressing cells which was required for ErbB2-mediated STAT3 activation and p21Cip1 increase. Treatment of ErbB2 over-expressing resistant cells with STAT3 inhibitor peptides sensitized the cells to Taxol. In addition to classical signal transduction pathways, I identified a novel ErbB2 mediated regulatory mechanism of p21Cip1. I found that a nuclear ErbB2 and STAT3 complex binds directly to the p21Cip1 promoter offering a non-classical mechanism of p21Cip1 promoter regulation. These data suggest that ErbB2 over-expression can confer Taxol resistance of breast cancer cells by transcriptional upregulation of p21 Cip1 via activation of STAT3 by Src kinase and also by cooperation with nuclear ErbB2. The data suggest a potential clinical mechanism for STAT3 inhibitors in sensitizing ErbB2 over-expressing breast cancers to Taxol. ^

Participation of the nuclear cap binding complex in pre-mRNA 3′ processing

Communication between the 5′ and 3′ ends is a common feature of several aspects of eukaryotic mRNA metabolism. In the nucleus, the pre-mRNA 5′ end is bound by the nuclear cap binding complex (CBC). This RNA–protein complex plays an active role in both splicing and RNA export. We provide evidence for participation of CBC in the processing of the 3′ end of the message. Depletion of CBC from HeLa cell nuclear extract strongly reduced the endonucleolytic cleavage step of the cleavage and polyadenylation process. Cleavage was restored by addition of recombinant CBC. CBC depletion was found to reduce the stability of poly(A) site cleavage complexes formed in nuclear extract. We also provide evidence that the communication between the 5′ and 3′ ends of the pre-mRNA during processing is mediated by the physical association of the CBC/cap complex with 3′ processing factors bound at the poly(A) site. These observations, along with previous data on the function of CBC in splicing, illustrate the key role played by CBC in pre-mRNA recognition and processing. The data provides further support for the hypothesis that pre-mRNAs and mRNAs may exist and be functional in the form of “closed-loops,” due to interactions between factors bound at their 5′ and 3′ ends.

Expansion of a CUG trinucleotide repeat in the 3′ untranslated region of myotonic dystrophy protein kinase transcripts results in nuclear retention of transcripts

Expansion of a CTG trinucleotide repeat in the 3′ untranslated region (UTR) of DMPK, the gene encoding myotonic dystrophy protein kinase, induces the dominantly inherited neuromuscular disorder myotonic dystrophy (DM). Transcripts containing the expanded trinucleotide are abundant in differentiated cultured myoblasts, and they are spliced and polyadenylylated normally. However, mutant transcripts never reach the cytoplasm in these nonmitotic cells; instead, they form stable clusters that are tightly linked to the nuclear matrix, which can prevent effective biochemical purification of these transcripts. In DM patients, reduced DMPK protein levels, consequent to nuclear retention of mutant transcripts, are probably a cause of disease development. Formation of nuclear foci is a novel mechanism for preventing transcript export and effecting a loss of gene function.

Splicing is required for rapid and efficient mRNA export in metazoans

Pre-mRNA splicing is among the last known nuclear events before export of mature mRNA to the cytoplasm. At present, it is not known whether splicing and mRNA export are biochemically coupled processes. In this study, we have injected pre-mRNAs containing a single intron or the same mRNAs lacking an intron (Δi-mRNAs) into Xenopus oocyte nuclei. We find that the spliced mRNAs are exported much more rapidly and efficiently than the identical Δi-mRNAs. Moreover, competition studies using excess Δi-mRNA indicate that different factor(s) are involved in the inefficient export of Δi-mRNA vs. the efficient export of spliced mRNA. Consistent with this conclusion, spliced mRNA and Δi-mRNA, though identical in sequence, are assembled into different messenger ribonucleoprotein particles (mRNP) in vitro. Strikingly, the mRNA in the spliced mRNP, but not in the Δi-mRNP, is exported rapidly and efficiently. We conclude that splicing generates a specific nucleoprotein complex that targets mRNA for export. Our results, revealing a link between splicing and efficient mRNA export, may explain the reports that an intron is required for efficient expression of many protein-coding genes in metazoans.

A multimeric complex and the nuclear targeting of the Drosophila Rel protein Dorsal

The intracellular part of the Rel signal transduction pathway in Drosophila is encoded by Toll, tube, pelle, dorsal, and cactus, and it functions to form the dorsal–ventral axis in the Drosophila embryo. Upon activation of the transmembrane receptor Toll, Dorsal dissociates from its cytoplasmic inhibitor Cactus and enters the nucleus. Tube and Pelle are required to relay the signal from Toll to the Dorsal–Cactus complex. In a yeast two-hybrid assay, we found that both Tube and Pelle interact with Dorsal. We confirmed these interactions in an in vitro binding assay. Tube interacts with Dorsal via its C-terminal domain, whereas full-length Pelle is required for Dorsal binding. Tube and Pelle bind Dorsal in the N-terminal domain 1 of the Dorsal Rel homology region rather than at the Cactus binding site. Domain 1 has been found to be necessary for Dorsal nuclear targeting. Genetic experiments indicate that Tube–Dorsal interaction is necessary for normal signal transduction. We propose a model in which Tube, Pelle, Cactus, and Dorsal form a multimeric complex that represents an essential aspect of signal transduction.