Analysis of radiation-induced bystander effects using high content screening

When cells are exposed to (ionising) radiation there is a rapid phosphorylation of a minor nucleosomal histone protein, H2AX, at the sites where double stranded breaks (DSB) occur. This phosphorylation is one of the earliest events in the repair cascade and extends over several mega base pairs surrounding the break. Nowadays it is generally accepted that the formation of γH2AX functions as a signal enhancer. Using immuno histochemistry we can visualise this phosphorylation as foci in the nucleus, where each foci represents a DSB [1]. For our research we use normal human primary fibroblasts (NHDF’s) to study the so called radiation-induced bystander effects which refer to the responses induced in non-irradiated cells, when neighbouring cells are irradiated. Although the exact pathways of transmission are yet to be determined, studies have shown that gap junction-mediated transport and secretion of soluble extracellular factors play an important role [2]. To exclude variation we first tried synchronisation of the fibroblasts using nocodazole or aphidicolin. Our attempts did not produce the desired synchronisation level. In addition, recent reports doubt the effectiveness of these products in cell synchronisation [3]. To resolve this problem, we used high content screening of cells together with specific cell cycle markers. One of these markers is 5-bromo-2-deoxyuridine (BrdU). BrdU, a synthetic nucleoside, is an analogue of thymidine that can be incorporated in replicating cells and specifically label S-phases [4]. Cells are cultured on membrane inserts, with a pore size of 0,4µm allowing soluble factors to pass but preventing the cells to interchange. These cells are irradiated with different doses and subsequently placed together with NHDF that are grown on cover glasses (see figure 1). Depending on the objectives BrdU is added 20-40 minutes before fixation. We found a differential pattern for γH2AX that we could specifically link to the cell cycle. During the S phase γH2AX is significantly more induced than during other phases of the cell cycle (see figure 2). This is probably due to the increased vulnerability caused by the unwinding of DNA during replication. 1. ¬¬S.H.Macphail, J.P.Banath, T.Y.Yu, E.H.Chu, H.Lambur, P.L.Olive, Int.J.Radiat.Biol. 79 (2003) P. 351-358. 2. H.Yang, N.Asaad, K.D.Held, Oncogene 24 (2005) p. 2096-2103. 3. S.Cooper, G.Iyer, M.Tarquini, P.Bissett, Cell Tissue Res. 324 (2006) p.237-242. 4. R.T.O'Keefe, S.C.Henderson, D.L.Spector, J.Cell Biol. 116 (1992) p.1095-1110

The Female Characters of Ernest Hemingway’s “The Last Good Country”

　　The three primary female characters of Ernest Hemingway’s unfinished story “The Last Good Country” are indispensable in aiding the male protagonist in his attempts at defying and subverting a malignantly depicted authority. In doing so, they defy and subvert authority themselves; the behavior depicted includes direct female defiance of traditional male authority figures. These strong and beneficent female characters are therefore directly at variance with the assertion by some feminist scholars since the 1970s that Hemingway women are misogynist stereotypes, an argument disputed in other research as well

Experience Implementing a Performant Category-Theory Library in Coq

We describe our experience implementing a broad category-theory library in Coq. Category theory and computational performance are not usually mentioned in the same breath, but we have needed substantial engineering effort to teach Coq to cope with large categorical constructions without slowing proof script processing unacceptably. In this paper, we share the lessons we have learned about how to represent very abstract mathematical objects and arguments in Coq and how future proof assistants might be designed to better support such reasoning. One particular encoding trick to which we draw attention allows category-theoretic arguments involving duality to be internalized in Coq's logic with definitional equality. Ours may be the largest Coq development to date that uses the relatively new Coq version developed by homotopy type theorists, and we reflect on which new features were especially helpful.Comment: The final publication will be available at link.springer.com. This version includes a full bibliography which does not fit in the Springer version; other than the more complete references, this is the version submitted as a final copy to ITP 201

Cranial anatomy and taxonomy of Dolichorhynchops bonneri new combination, a polycotylid (Sauropterygia: Plesiosauria) from the Pierre Shale of Wyoming and South Dakota

The taxonomic identity of two well-preserved polycotylid plesiosaur skeletons from the Pierre Shale of far northern Wyoming and southern South Dakota has been controversial since their discovery. Originally referred to Dolichorhynchops osborni, the material was almost immediately christened Trinacromerum bonneri Adams 1997; more recently the material has been referred toPolycotylus. Recent preparation of the well-preserved skull of one specimen permits detailed examination of the cranial morphology of this animal for the first time, and allows for its inclusion in a cladistic analysis of the Polycotylidae. This analysis reveals a stable sister-taxon relationship with Dolichorhynchops osborni. However, the taxon possesses a bewildering mosaic of character states, superficially resembling Polycotylus in overall size and tooth morphology and Trinacromerumin details of the palate and lower jaw, while sharing several critical synapomorphies withDolichorhynchops osborni. This wide-ranging homoplasy among characters previously diagnostic among polycotylid genera challenges the alpha taxonomy of forms from the western interior seaway. The conservative taxonomic course of referring the species T. bonneri to Dolichorhynchopsis followed here, but a species-level review is necessary. Lastly, material described here reveals important new information concerning the polycotylid orbital region and lower jaw, allowing confident reconstructions of these controversial regions for the first time