Poly(ADP-Ribosyl)ation affects stabilization of CHE-1 protein in response to DNA damage

Post-translation modifications play a crucial role in coordinating the cellular response to DNA damage. Double strand DNA breaks (DSBs) trigger the activation of ATM and Chk2 kinases, which represent the primary transducers in the signalling cascade. Among the high number of phosphorylated proteins, our attention was focused on Che-1, a novel ATM and Chk2 substrate whose role in DNA damage response has been recently shown. Phosphorylated Che-1 accumulates and promotes transcription of p53 and p53-responsive genes, which are critical for the maintenance of G2 arrest and for DNA repair processes . Poly(ADP-ribosyl)ation is a post-translational modification that shows an emerging role in the signal transduction to the DDR machinery. Poly(ADP-ribose) polymerase 1 (PARP-1), the main enzyme involved in this modification, is recruited on DNA lesions and catalyzes the synthesis of poly(ADP-ribose) polymers (PAR) on itself and on target proteins. In particular, a recent work demonstrated that PAR synthesis at DSBs sites is necessary to recruit ATM kinase, which can interact non-covalently with PAR. In this study we showed that poly(ADP-ribosyl)ation, beyond phosphorylation, is involved in the regulation of Che-1 stabilization following DNA damage. We demonstrated that Che-1 accumulation upon doxorubicin treatment is reduced after inhibition of PARP activity in HCT116 cells and in PARP-1 knock-out or silenced cells. In accordance, impairment in Che-1 accumulation by PARP inhibition reduced Che-1 occupancy at p21 promoter and affected the expression of the corresponding gene. Epistasis experiments showed that the effect of poly(ADP-ribosyl)ation on Che-1 stabilization is independent from ATM kinase activity. Indeed we demonstrated that Che-1 protein co-immunoprecipitates with ADP-ribose polymers and that PARP-1 directly interacts with Che-1, promoting its modification in vitro and in vivo. Altogether, these findings suggest that poly(ADP-ribosyl)ation of Che-1 represents a mechanism enabling the precise control over the level of Che-1 protein in response to DNA damage

Modelling of bubble nucleation in trachy-phonolitic magmas: implications for the dynamics of ash-rich eruptions

Nucleation of water gas bubbles in trachyphonolitic magmatic melts has been investigated integrating theory and numerical modelling with decompression experiments and analysis of natural ash samples of explosive eruptions. Bubble nucleation, considered the natural response of magmas to decompression, is strongly dictated by the gas-melt surface tension. Here, I use an integrated approach to quantify the role of the surface tension in the nucleation process combining high pressure - high temperature nucleation experiments with a numerical modelling based on the gradient theory (Cahn and Hilliard, 1959). This theory, successfully applied in several studies of industrial polymers (Poser and Sanchez, 1981; Harrison et al., 1999; Kahl and Enders, 2000; Enders et al., 2005) was never been used before to study systems of volcanological interest. I show that surface tension is far to be a constant, but it decreases with in- creasing nucleation pressure (i.e. the confining pressure). Entering the values of surface tension into the classical theory of nucleation, I obtain a variable supersaturation pressure triggering nucleation. The decreasing value of the gas-melt surface tension with increasing pressure, facilitate bubble nucleation at high pressure, thus enhancing the explosivity of eruptive events from deeper reservoirs. Instead, the hindered nucleation at relatively low pressure, due to high bubble surface tension, implies that the generation of explosive eruptions from shallow reservoirs requires high decompressions. Finally the vesiculation, in terms of nucleation and growth, of natural samples of ash-rich eruptions has been studied by applying a novel technique able to take 3D measurements of bubbles preserved on ash particle’s surface. The Bubble Size Distributions (BSD), together with the field evidence, suggest that the ash production in these ash-rich eruptions, rather than to magma-water explosive interaction, is related to the high decompression necessary to nucleate bubbles in a shallow reservoir

Azathioprine-Induced Peripheral T Cell Apoptosis And Drug Response In Patients With Crohn’s Disease

Background and Aim: the long time interval for a trial of thioupurine therapy and the potential side effects in spite of the proven efficacy, do not encourage their use as early therapeutic option in Crohn’s Disease (CD). The development of tests predictive of responsiveness represents a major attempt in the clinical management of CD patients. Azathioprine (AZA) is able to induce apoptosis of T cells; therefore we analyzed the “in vitro” thiopurine-induced T cells apoptosis in a group of CD patients with known response to a previous treatment with AZA. Methods: peripheral CD4+ T cells from 16 CD patients were stimulated with antiCD3/CD28 mAbs in the presence or absence of AZA or 6-MP or 6-thioguanine; apoptosis was assessed using Annexin V staining. Results: Apoptosis stimulation index (% of apoptotic cells in the presence of thiopurine / % of apoptotic cells in their absence) was significantly lower in non responder when compared to responder patients (1.46 (0.97-1.8) vs. 2.19 (1.58-2.65) median (range), respectively; p=0.002 by Mann Whitney test). Conclusions: evaluation of apoptosis stimulation index of peripheral CD4+T cell induced by AZA might represent a parameter useful for a proper selection of CD patients candidate to thiopurine treatment

Methylation status of Dnmt1 promoter depends on poly(ADP-ribosy)lation

Research is focused on CpG islands and on the mechanism that poly(ADP-ribosyl)ation uses to defend the unmethylated state of these important DNA sequences which are located in the promoter regions of the housekeeping genes having a role of transcription regulators. Data here reported show that inhibition of PARP activity allows the diffuse insertion of methyl groups onto some CpG islands and in particular on the CpG island which is located in the promoter region of Dnmt1 gene. Hence, following inhibition of PARPs activity, this promoter loses its protection against methylation becoming silenced through methylation as shown by analyses with Methylation Sensitive PCR (MS-PCR) and sequencing after bisulphite treatment. Analyses of Western Blotting, RT-PCR and Real-time RT-PCR confirm that the gene has undergone silencing. The role of ADP-ribose polymers in silencing Dnmt1 has been demonstrated by additional experiments in which overexpression of poly(ADP-ribose) glycohydrolase leads to reduction of ADP-ribose polymers in nuclei associated to a sharp decrease of Dnmt1 level respect to control. A parallel genome-wide methyl-sensitive restriction assay demonstrates that the variation of Dnmt1 level is followed by a bimodal alteration of DNA methylation pattern. In fact, the inhibition of poly(ADP-ribosyl)ation initially causes an increase in methyl-group insertion onto DNA while this phenomenon is reversed after prolonged treatments and demethylation is detected within Alu sequences. Considering the important role played by Dnmt1 in the epigenetic scenario, these data lead us to think about what happens in tumor cells where both anomalous methylation of some CpG islands and diffuse hypomethylation are present. These findings open up a new path into epigenetic research in tumors. What is remarkable is that the demethylated pattern found in Alu sequences after treatment of cells with 3-ABA for 96 hours is very similar to the one found on DNA from cells treated with 5-AZA for the same time. The discovery of a DNA demethylating activity dependent on the use of inhibitors of poly(ADP-ribosyl)ation process increases the knowledge of mechanism by which these inhibitors enhance the cytotoxicity of other anticancer agents

Inequalities with angular integrability and applications

We prove an extension of the Stein-Weiss weighted estimates for fractional integrals, in the context of Lp spaces with different integrability properties in the radial and the angular direction. In this way, the classical estimates can be unified with their improved radial versions. A number of consequences are obtained: in particular we deduce precised versions of weighted Sobolev embeddings, Caffarelli-Kohn-Nirenberg estimates, and Strichartz estimates for the wave equation, which extend the radial improvements to the case of arbitrary functions. Then we apply this technology in order to give new a priori assumptions on weak solutions of the Navier-Stokes equation so as to be able to conclude that they are smooth. The regularity criteria are given in terms of mixed radial-angular weighted Lebesgue space norms.Comment: Phd Thesis at University Sapienza, advisor professor Piero D'Ancon

Functional evaluation of hepatic parenchyma with dynamic tests (Indocyanine green and Hippurate ratio) in patients with HCC

Our aim was to assess hepatic functionality in patients with HCC and candidate for liver resection. We used dynamic tests of liver functionality Indocyanine green (ICG) and Hippurate ratio, for the evaluation of surgical risk and postoperative clinical course. The clinical and predictive value of the tests has been compared with each other and with routine tests. Tests have been performed on 11 patients with HCC, in 8 before surgery and after 6 months, in 1 before TACE and after 6 months. The results showed a significative correlation with the endpoints: survival, liver failure, general condition of the patients, and parallelism in the clinical assessment of patients. ICG and Hippurate ratio are useful in patients undergoing liver resection and in the follow up

An upper bound for the genus of a curve without points of small degree

In this paper I prove that for any prime $p$ there is a constant $C_p>0$ such that for any $n>0$ and for any $p$-power $q$ there is a smooth, projective, absolutely irreducible curve over $\mathbb{F}_q$ of genus $g\leq C_p q^n$ without points of degree smaller than $n$.Comment: This is part of a Phd thesis at Universit\`a 'Sapienza' of Rom

Transcriptional landscape of neuronal and cancer stem cells

Tumor mass is composed by heterogeneous cell population including a subset of “cancer stem cells” (CSC). Oncogenic signals foster CSC by transforming tissue stem cells or by reprogramming progenitor/differentiated cells towards stemness. Thus, CSC share features with cancer and stem cells (e.g. self-renewal, hierarchical developmental program leading to differentiated cells, epithelial/mesenchimal transition) and these latter are maintained by the constitutive activation of stemness-promoting signals. CSC could trigger tumor formation, drive to resistance to conventional therapeutics and underlie patients’ relapse. Indeed, stem cell signatures have been associated with poor prognosis in various. This background makes the identification of CSC molecular features mandatory to highlight the survival inner working and to design novel CSC specific therapeutic strategies. Medulloblastoma (MB) is the most common childhood malignant brain tumor and a leading cause of cancerrelated morbidity and mortality. Current multimodal therapies are effective in about 50% of patients but often cause long-term side effects, i.e. developmental, neurological, neuroendocrine and psychosocial deficits (Northcott PA Nature Rev cancer 2012). For many years, MB treated as a single tumor entity despite the divergent tumor histology, patients’ outcome and drug sensitivity, and also by the diversity of the stem cell of origin. Very recently the scenario of human MB has dramatically changed since its heterogeneous biology has been addressed by high-throughput gene expression analysis (oligonucleotide microarrays) or by the powerful genomic next-generation sequencing. These led to the identification of four tumor subgroups (WNT, SHH, Group 3 and Group 4) uncovering the existence of a highly diverse mutational spectra and gene expression. However a quantitative approach has not yet been applied to the transcriptional landscape of Medulloblastoma stem cells (MbSC) through RNA Next Generation Sequencing (RNA-Seq) technology. This is a relevant issue, since RNA-Seq is able to interrogate the genome wide global transcriptome including new transcripts, alternative spliced isoforms and non-coding RNAs. Lower rhombic lip progenitors of the dorsal brainstem are considered the trigger cells in WNT tumors; in SHH subgroup initiation cells are Prominin1+ CD15+ stem cells from the subventricular zone requiring the commitment to Math1+ granule cell progenitors [GCP] of the external granule cell layer [EGL]; while Math1+ or Math1- EGL-GCP or Prominin1+/lineage-negative stem cells sustain the MYC driven Group 3. MbSC derived from SHH tumors and postnatal normal cerebellar stem cells (NcSC) have been reported to share several features. A key signal for both of them is Hedgehog. Furthermore, both NcSC and MbSC display up-regulation of stemness genes (e.g Sox2, Nestin, Nanog, Prom1). Finally, constitutive activation of the Shh pathway by conditional deletion of Ptch1 inhibitory receptor in NcSC, promote medulloblastoma in vivo, producing a mouse model of the human SHH tumor. Acquisition of stemness features may therefore represent the first step of oncogenic conversion. Cooperation with additional oncogenic signals is however needed to enhance MbSC tumorigenicity. In order to understand the MbSCs transcriptional programs, we analyze by RNA-Seq, MbSC derived from Ptch1+/- tumors (Ptch1+/- MbSC). This choice, of a genetically determined model of MB, has allowed us to work with Ptch1+/- MbSC together with appropriate NcSC counterpart, and to analyze biological replicates doing statistical analysis. We identify a number of transcripts, annotated ones, novel isoforms, and long non-coding RNAs, characterizing MbSC and/or NcSC. Some of these genes control stemness or are cancer related and conserved in human medulloblastomas. Interestingly a subset of them, belonging to cell stress response, are of prognostic relevance being significantly related to clinical outcome. Correlation of genes expression characterizing MbSC with survival information from our human medulloblastomas database further demonstrates the significance of these findings. Our data suggest that the modulation of normal and cancer stem cell functions observed in vitro is effective in dissecting the transcriptional programs underlying the in vivo behavior of human medulloblastomas