AN ANALYSIS OF THE DNA DAMAGE INDUCED BY NICKEL COMPOUNDS IN CHINESE HAMSTER OVARY CELLS (CARCINOGENESIS, HETEROCHROMATIN, CYTOTOXICITY, REPLICATION, PROTEIN CROSSLINKING)

The carcinogenic activity of water-insoluble crystalline nickel sulfide requires phagocytosis and lysosome-mediated intracellular dissolution of the particles to yield Ni('2+). This study investigated the extent and nature of the DNA damage in Chinese hamster ovary cells treated with various nickel compounds using the technique of alkaline elution. Crystalline NiS and water-soluble NiCl(,2) induced single strand breaks that were repaired quickly and DNA-protein crosslinks that persisted up to 24 hr after exposure to nickel. The induction of single strand breaks was concentration dependent at both noncytotoxic and lethal amounts of nickel. The induction of DNA-protein crosslinks was concentration dependent but was absent at lethal amounts of nickel. The cytoplasmic and nuclear uptake of nickel was concentration dependent even at the toxic level of nickel. However, the induction of DNA-protein crosslinks by nickel required active cell cycling and occurred predominantly in mid-late S phase of the cell cycle, suggesting that the lethal amounts of nickel inhibited DNA-protein crosslinking by inhibiting active cell cycling. Since the DNA-protein crosslinking induced by nickel was resistant to DNA repair, the nature of this lesion was investigated using various methods of DNA isolation and chromatin fractionation in combination with SDS-polyacrylamide gel electrophoresis. High molecular weight, non-histone chromosomal proteins and possibly histone 1 were preferentially crosslinked to DNA by nickel. The crosslinked proteins were concentrated in a magnesium-insoluble fraction of sonicated chromatin (5% of the total) that was similar to heterochromatin in solubility and protein composition. Alterations in DNA structure and function, brought about by the effect of nickel on protein-DNA interactions, may be related to the carcinogenicity of nickel compounds. ^

PREMATURE CONDENSATION OF SPERMATOGENIC CELL CHROMOSOMES

The technique of premature chromosome condensation (PCC) has been used primarily to study interphase chromosomes of somatic cells. In this study, mitotic cells were fused to cells from the mouse testes to examine the chromosomes of germ cells. The testes contain various types of cells, both germinal and nongerminal. In these initial studies, four types of PCC morphologies were observed. Chromosome morphology of the PCC and labeling experiments demonstrated the mouse cell origin of various PCC. Attempts were next made to determine the cell types producing the PCC. Spermatogonia, diplotene spermatocytes, secondary spermatocytes and round spermatids are proposed to be the origin of the PCC morphologies. Some PCC could be banded by G and C banding techniques and the mouse chromosomes identified.^ Studies were subsequently undertaken to evaluate this technique as a method of evaluating damage to germ cells. Testicular cells from irradiated mice were fused to mitotic cells and the PCC examined. Both round spermatids and secondary spermatocytes exhibited chromosome damage in the form of chromatid breaks. A linear correlation was found between the dose of irradiation and the number of breaks per cell. This technique may develop into a useful method for evaluating the clastogenic effect of agents on the germ cells. ^

Transition and Justice: An Introduction

Since the end of the Cold War, political new beginnings have increasingly been linked to questions of transitional justice. The contributions to this collection examine a series of cases from across the African continent where peaceful ‘new beginnings’ have been declared after periods of violence and where transitional justice institutions played a role in defining justice and the new socio-political order. Three issues seem to be crucial to the understanding of transitional justice in the context of wider social debates on justice and political change: the problem of ‘new beginnings’, of finding a foundation for that which explicitly breaks with the past; the discrepancies between lofty promises and the messy realities of transitional justice in action; and the dialectic between logics of the exception and the ordinary, employed to legitimize or resist transitional justice mechanisms. These are the particular focus of this Introduction.

Control of human behavior, mental processes and consciousness : essays in honor of the 60th birthday of August Flammer

In this book, an international group of leading scientists present perspectives on the control of human behavior, awareness, consciousness, and the meaning and function of perceived control or self-efficacy in people's lives. The book breaks down the barriers between subdisciplines, and thus constitutes an occasion to reflect on various facets of control in human life. Each expert reviews his or her field through the lens of perceived control and shows how these insights can be applied in practice.

Characterization of the potential role for RNA-binding protein FUS/TLS in DNA damage response: A quantitative proteomic approach

FUS/TLS (fused in sarcoma/translocated in liposarcoma) is a ubiquitously expressed RNA-binding protein of the hnRNP family, that has been discovered as fused to transcription factors, through chromosomal translocations, in several human sarcomas and found in protein aggregates in neurons of patients with an inherited form of Amyotrophic Lateral Sclerosis (ALS) [1]. To date, FUS/TLS has been implicated in a variety of cellular processes such as gene expression control, transcriptional regulation, pre-mRNA splicing and miRNA processing [2]. In addition, some evidences link FUS/TLS to genome stability control and DNA damage response. In fact, mice lacking FUS/TLS are hypersensitive to ionizing radiation (IR) and show high levels of chromosome instability and in response to double-strand breaks, FUS/TLS gets phosphorylated by the protein kinase ATM [3,4,5]. Furthermore, the inducible depletion of FUS/TLS in a neuroblastoma cell line (SH-SY5Y FUS/TLS TET-off iKD) subjected to genotoxic stress (IR) resulted in an increased phosphorylation of γH2AX respect to control cells, suggesting an higher activation of the DNA damage response. The study aims to investigate the specific role of FUS/TLS in DNA damage response through the characterization of the proteomic profile of SH-SY5Y FUS/TLS iKD cells subjected to DNA damage stress, by mass spectrometry-based quantitative proteomics (e.g. SILAC). Preliminary results of mass spectrometric identification of FUS/TLS interacting proteins in HEK293 cells, expressing a recombinant flag-tagged FUS/TLS protein, highlighted the interactions with several proteins involved in DNA damage response, such as DNA-PK, XRCC-5/-6, and ERCC-6, raising the possibilities that FUS/TLS is involved in this pathway, even thou its exact role still need to be addressed.

Characterization of the potential role for RNA-binding protein FUS in DNA damage response: A quantitative proteomic approach

FUS/TLS (fused in sarcoma/translocated in liposarcoma) is a ubiquitously expressed protein of the hnRNP family, that has been discovered as fused to transcription factors in several human sarcomas and found in protein aggregates in neurons of patients with an inherited form of Amyotrophic Lateral Sclerosis [Vance C. et al., 2009]. FUS is a 53 kDa nuclear protein that contains structural domains, such as a RNA Recognition Motif (RRM) and a zinc finger motif, that give to FUS the ability to bind to both RNA and DNA sequences. It has been implicated in a variety of cellular processes, such as pre-mRNA splicing, miRNA processing, gene expression control and transcriptional regulation [Fiesel FC. and Kahle PJ., 2011]. Moreover, some evidences link FUS to genome stability control and DNA damage response: mice lacking FUS are hypersensitive to ionizing radiation (IR) and show high levels of chromosome instability and, in response to double-strand breaks, FUS is phosphorylated by the protein kinase ATM [Kuroda M. et al., 2000; Hicks GG. et al., 2000; Gardiner M. et al., 2008]. Furthermore, preliminary results of mass spectrometric identification of FUS interacting proteins in HEK293 cells, expressing a recombinant flag-tagged FUS protein, highlighted the interactions with proteins involved in DNA damage response, such as DNA-PK, XRCC-5/-6, and ERCC-6, raising the possibilities that FUS is involved in this pathway, even though its role still needs to be clarified. This study aims to investigate the biological roles of FUS in human cells and in particular the putative role in DNA damage response through the characterization of the proteomic profile of the neuroblastoma cell line SH-SY5Y upon FUS inducible depletion, by a quantitative proteomic approach. The SH-SY5Y cell line that will be used in this study expresses, in presence of tetracycline, a shRNA that targets FUS mRNA, leading to FUS protein depletion (SH-SY5Y FUS iKD cells). To quantify changes in proteins expression levels a SILAC strategy (Stable Isotope Labeling by Amino acids in Cell culture) will be conducted on SH-SY5Y FUS iKD cells and a control SH-SY5Y cell line (that expresses a mock shRNA) and the relative changes in proteins levels will be evaluated after five and seven days upon FUS depletion, by nanoliquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS) and bioinformatics analysis. Preliminary experiments demonstrated that the SH-SY5Y FUS iKD cells, when subjected to genotoxic stress (high dose of IR), upon inducible depletion of FUS, showed a increased phosphorylation of gH2AX with respect to control cells, suggesting an higher activation of the DNA damage response.

The quiet eye and tasks demands: Do tougher shots need a quieter eye?

The Quiet Eye (QE; Vickers 1996) has been shown to underpin successful performance, differentiating both expertise (inter-individual) and proficiency (intra-individual), with experts and successful attempts characterised by longer QE durations. The QE is proposed to reflect the time needed to organise and fine tune the parameters of movement (e.g. force and direction). In order to examine this prediction and build upon previous research we experimentally manipulated the difficulty of a golf putting task; we hypothesised that if the QE is related to motor programming then a more difficult task should be associated with longer QE durations. 33 golfers (M age= 21.16, SD= 3.98) with an average handicap of 6.5 (SD= 6.02) performed putts in 4 conditions of increasing difficulty. We manipulated the length of the golf putt (short-4ft, long-8ft) and the contact point of the putter head (large-1.7cm, small-0.5cm,) giving increasingly difficult putting conditions of short-large [1], short-small [2], long-large [3] and long-small [4]. We measured performance (radial error from hole in cm) and QE (in ms) for 10 putts in each condition. A repeated measures ANOVA was performed on the performance and QE data. The performance data suggest that we were successful in increasing the difficulty of the task (F (3,93) = 26.46. p = .000), with the best performance in condition [1] (8.57cm), followed by [2] (9.10cm) followed by [3] (16.11cm) and finally the worst performance was in condition [4] (23.40cm). The QE data suggest that, in keeping with our hypothesis, the QE was lengthened as task difficulty increased (F (3,87) = 11.91, p = .043). The QE was shortest in condition [1] (1787.85ms) and increased to condition [2] (1939.78ms) and condition [3] (2076.51ms), with the longest QE in condition [4] (2164.08ms). More detailed analysis of the QE reveal that it was the proportion of the QE that occurred before movement initiation (pre-QE) which increased with shot difficulty, rather than the proportion that occurred during the swing (Online-QE; see Vine et al., 2013). Results support the notion that more complex tasks are associated with a longer QE duration, specifically participants appear to spend longer fixating the target prior to movement. This likely reflects the time needed to process visual information gathered in a pre-performance routine, to inhibit external distraction, and to pre-programme the increasingly difficult parameters of the movement. Vickers, J.N. (1996). Visual control when aiming at a far target. Journal of Experimental Psychology: Human Perception and Performance, 22, 342-354. Vine, S.J. et al. (2013). Quiet eye and choking: Online control breaks down at the point of performance failure. Medicine and Science in Sports and Exercise, 45, 1988-1994.