Sinumerik 840D tools for programming of CNC machines

Diplomová práce uvádí historicky vývoj a současnost CNC obráběcích strojů a jejich řídicích systémů. Také v práci jsou popsané základy a metodiky programování, pomocí kterých může byt realizována tvorba NC programu v ŘS Sinumerik 840D. Druha část práce zahrnuje zpracování výrobního postupu součásti (skříň převodovky) a NC programu. Ten program je vytvořen v dílensky orientovaném prostředí ShopMill. Ověření programu bylo zajištěno pomocí modulu simulace řídicího systému.Master’s thesis presents the history and the present-day state of CNC machines and their control systems. This thesis describes both the basics and the advanced methods of programming which can provide the ability to create NC program for Sinumerik 840D to operate. The second part of the thesis deals with the development of the technology to machine the part (the frame of the reduction gear) and corresponding NC program. This program is created by means of the work-step programming (ShopMill). The final verification of the program was provided by means of simulation component of the control system.

Genome-wide DNA sampling by Ago nuclease from the cyanobacterium Synechococcus elongatus

Members of the conserved Argonaute (Ago) protein family provide defense against invading nucleic acids in eukaryotes in the process of RNA interference. Many prokaryotes also contain Ago proteins that are predicted to be active nucleases, however, their functional activities in host cells remain poorly understood. Here, we characterize the in vitro and in vivo properties of the SeAgo protein from the mesophilic cyanobacterium Synechococcus elongatus. We show that SeAgo is a DNA-guided nuclease preferentially acting on single-stranded DNA targets, with nonspecific guide-independent activity observed for double-stranded substrates. The SeAgo gene is steadily expressed in S. elongatus, however, its deletion or overexpression does not change the kinetics of cell growth. When purified from its host cells or from heterologous E. coli, SeAgo is loaded with small guide DNAs whose formation depends on the endonuclease activity of the argonaute protein. SeAgo co-purifies with SSB proteins suggesting that they may also be involved in DNA processing. The SeAgo-associated small DNAs are derived from diverse genomic locations, with certain enrichment for the proposed sites of chromosomal replication initiation and termination, but show no preference for an endogenous plasmid. Therefore, promiscuous genome sampling by SeAgo does not have great effects on cell physiology and plasmid maintenance

The Impact of External Reference Pricing within and across Countries

An assessment of the impact of external reference pricing (ERP) systems on important health system goals such as availability, affordability and diffusion/utilisation of pharmaceuticals; and an analysis of the impact that ERP systems have at the domestic and international levels, particularly considering their likely spillover effects

Genome-wide DNA sampling by Ago nuclease from the cyanobacterium Synechococcus elongatus

Members of the conserved Argonaute (Ago) protein family provide defense against invading nucleic acids in eukaryotes in the process of RNA interference. Many prokaryotes also contain Ago proteins that are predicted to be active nucleases, however, their functional activities in host cells remain poorly understood. Here, we characterize the in vitro and in vivo properties of the SeAgo protein from the mesophilic cyanobacterium Synechococcus elongatus. We show that SeAgo is a DNA-guided nuclease preferentially acting on single-stranded DNA targets, with nonspecific guide-independent activity observed for double-stranded substrates. The SeAgo gene is steadily expressed in S. elongatus, however, its deletion or overexpression does not change the kinetics of cell growth. When purified from its host cells or from heterologous E. coli, SeAgo is loaded with small guide DNAs whose formation depends on the endonuclease activity of the argonaute protein. SeAgo co-purifies with SSB proteins suggesting that they may also be involved in DNA processing. The SeAgo-associated small DNAs are derived from diverse genomic locations, with certain enrichment for the proposed sites of chromosomal replication initiation and termination, but show no preference for an endogenous plasmid. Therefore, promiscuous genome sampling by SeAgo does not have great effects on cell physiology and plasmid maintenance

Additional data for article "Genome-wide DNA sampling by Ago nuclease from the cyanobacterium Synechococcus elongatus"

Protein mass spectrometry data; small DNAs libraries sequencing data for article "Genome-wide DNA sampling by Ago nuclease from the cyanobacterium Synechococcus elongatus

Bacterial Argonaute Proteins Aid Cell Division in the Presence of Topoisomerase Inhibitors in Escherichia coli

Prokaryotic Argonaute (pAgo) proteins are guide-dependent nucleases that function in host defense against invaders. Recently, it was shown that TtAgo from Thermus thermophilus also participates in the completion of DNA replication by decatenating chromosomal DNA. Here, we show that two pAgos from cyanobacteria Synechococcus elongatus (SeAgo) and Limnothrix rosea (LrAgo) are active in heterologous Escherichia coli and aid cell division in the presence of the gyrase inhibitor ciprofloxacin, depending on the host double-strand break repair machinery. Both pAgos are preferentially loaded with small guide DNAs (smDNAs) derived from the sites of replication termination. Ciprofloxacin increases the amounts of smDNAs from the termination region and from the sites of genomic DNA cleavage by gyrase, suggesting that smDNA biogenesis depends on DNA replication and is stimulated by gyrase inhibition. Ciprofloxacin enhances asymmetry in the distribution of smDNAs around Chi sites, indicating that it induces double-strand breaks that serve as a source of smDNA during their processing by RecBCD. While active in E. coli, SeAgo does not protect its native host S. elongatus from ciprofloxacin. These results suggest that pAgo nucleases may help to complete replication of chromosomal DNA by promoting chromosome decatenation or participating in the processing of gyrase cleavage sites, and may switch their functional activities depending on the host species.IMPORTANCE Prokaryotic Argonautes (pAgos) are programmable nucleases with incompletely understood functions in vivo. In contrast to eukaryotic Argonautes, most studied pAgos recognize DNA targets. Recent studies suggested that pAgos can protect bacteria from invader DNA and counteract phage infection and may also have other functions including possible roles in DNA replication, repair, and gene regulation. Here, we have demonstrated that two cyanobacterial pAgos, SeAgo and LrAgo, can assist DNA replication and facilitate cell division in the presence of topoisomerase inhibitors in Escherichia coli. They are specifically loaded with small guide DNAs from the region of replication termination and protect the cells from the action of the gyrase inhibitor ciprofloxacin, suggesting that they help to complete DNA replication and/or repair gyrase-induced breaks. The results show that pAgo proteins may serve as a backup to topoisomerases under conditions unfavorable for DNA replication and may modulate the resistance of host bacterial strains to antibiotics.ISSN:2165-049

Successive Grinding and Polishing Effect on the Retained Austenite in the Surface of 42CrMo4 Steel

Low-alloy 42CrMo4 steels were studied by Fe-57 Mossbauer spectroscopy (MS), X-ray diffractometry (XRD), and Energy Dispersive X-ray Spectroscopy (EDS) measurements. The investigations were performed on metallographic samples, which were subjected to a series of successive grinding and polishing with a progressively finer grit. Conversion X-ray Mossbauer spectroscopy (CXMS) was used to determine the occurrence of austenite in steel samples. It is a unique method detecting the austenite content very sensitively. Six samples with different surface preparation were investigated, starting with 4.8% of austenite on an as-cut sample, and a large decrease in the retained austenite to 2.6% was observed after the first grinding of a hardened cut sample. Additionally, an unexpectedly large decrease in the austenite content to 2.3% was found due to the final polishing. A second time applied successive grinding and polishing of all samples resulted in identical austenite content determined by CXMS of approx. 5%, which proved the applicability of the CXMS method. Generally, the result calls attention to the importance of preparation of metallurgical samples by grinding and polishing where the results can vary significantly on the level of surface processing