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

A Cladistic Analysis and Taxonomic Revision of the Plesiosauria (Reptilia: Sauropterygia)

The Plesiosauria (Reptilia: Sauropterygia) is a group of Mesozoic marine reptiles known from abundant material, with specimens described from all continents. The group originated very near the Triassic–Jurassic boundary and persisted to the end- Cretaceous mass extinction. This study describes the results of a specimen-based cladistic study of the Plesiosauria, based on examination of 34 taxa scored for 166 morphological characters. The Pliosauroidea is found to by polyphyletic due to the inclusion of the Polycotylidae; this second clade is instead a member of the Plesiosauroidea, and thus more closely related to elasmosaurs than to other ‘pliosaurs’. Characters of body proportion such as neck length and head size are very labile, with the ‘pliosauromorph’ body plan evolving three times, while extremely long necks evolved in two clades. Characters from the entire skeleton support these relationships, although characters of the skull roof and palate are especially useful. Lastly, a new genus and species, Hauffiosaurus zanoni, is named based on German material of Toarcian age