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Ribonucleolytic resection is required for repair of strand displaced nonhomologous end-joining intermediates

Nonhomologous end-joining (NHEJ) pathways repair DNA double-strand breaks (DSBs) in eukaryotes and many prokaryotes, although it is not reported to operate in the third domain of life, archaea. Here, we describe a complete NHEJ complex, consisting of DNA ligase (Lig), polymerase (Pol), phosphoesterase (PE), and Ku from a mesophillic archaeon, Methanocella paludicola (Mpa). Mpa Lig has limited DNA nick-sealing activity but is efficient in ligating nicks containing a 3′ ribonucleotide. Mpa Pol preferentially incorporates nucleoside triphosphates onto a DNA primer strand, filling DNA gaps in annealed breaks. Mpa PE sequentially removes 3′ phosphates and ribonucleotides from primer strands, leaving a ligatable terminal 3′ monoribonucleotide. These proteins, together with the DNA end-binding protein Ku, form a functional NHEJ break-repair apparatus that is highly homologous to the bacterial complex. Although the major roles of Pol and Lig in break repair have been reported, PE’s function in NHEJ has remained obscure. We establish that PE is required for ribonucleolytic resection of RNA intermediates at annealed DSBs. Polymerase-catalyzed strand-displacement synthesis on DNA gaps can result in the formation of nonligatable NHEJ intermediates. The function of PE in NHEJ repair is to detect and remove inappropriately incorporated ribonucleotides or phosphates from 3′ ends of annealed DSBs to configure the termini for ligation. Thus, PE prevents the accumulation of abortive genotoxic DNA intermediates arising from strand displacement synthesis that otherwise would be refractory to repair

Structural and functional characterisation of the Nonhomologous End-­‐Joining proteins of the archaeon Methanocella Paludicola

Maintenance of the genome is essential for life to prosper. Regular insults to the genome are sustained by all cellular life and can foster genetic instability if left unrepaired. The most lethal genetic damage is a double strand break (DSB), the cleavage of the phosphate backbone on both strands of the DNA double helix. Two main pathways exist which provide mechanisms for coping with DSBs; precise repair utilising the identical sister chromatid as a template to recreate the broken segment (homologous recombination; HR), and direct fusion of the broken ends in the absence of an intact template (nonhomologous end joining; NHEJ). NHEJ was first characterised in eukaryotes, and an analogous system has been found to exist in bacteria during the past decade. The bacterial NHEJ pathway is composed of four key proteins; the DNA end binding Ku homodimer, a DNA Ligase, a DNA polymerase and a phosphoesterase (PE). The first results chapter of this thesis details the identification of an orthologous set of proteins in the archaeon Methanocella paludicola, and their subsequent isolation and characterisation. The second results chapter expands on the individual activities of the proteins by combining them, and asserting the ability of archaeal NHEJ to join discontinuous ends in vitro. The role of the PE has been unclear in the bacterial system, but in vitro assays described here suggest that the enzyme plays a role in processing NHEJ intermediates formed by the NHEJ polymerase. The PE is found to optimise repair intermediates for ligation, and to reverse potentially genotoxic DNA strand displacements. The final results chapter investigates the structural aspects of the archaeal NHEJ enzymes. Together these studies establish a functional NHEJ system in an archaeon for the first time, and expand our knowledge of the bacterial system by proposing a standard model of archaeo--‐prokaryotic NHEJ

Age-Related Differences in Auditory Processing as Assessed by Amplitude-Modulation Following Responses in Quiet and in Noise

Our knowledge of age-related changes in auditory processing in the central auditory system is limited, unlike the changes in the peripheral hearing organs which are more extensively studied. This study aims to further understanding of temporal processing in aging using non-invasive electrophysiological measurements in a rat model system. Amplitude modulation following responses (AMFRs) were assessed using sinusoidally amplitude modulated (SAM) tones presented to aged (92- to 95-weeks old) and young (9- to 12-weeks old) Fischer-344 rats. The modulation frequency and sound level were systematically varied, and the SAM stimuli were also presented simultaneously with wideband background noise at various levels. The overall shapes and cutoff frequencies of the AMFR temporal modulation transfer functions (tMTFs) were similar between young and aged animals. The fast Fourier transform (FFT) amplitudes of the aged animals were similar to the young in the 181–512 Hz modulation frequency range, but were significantly lower at most modulation frequencies above and below. There were no significant age-related differences in the nature of growth or FFT amplitudes with change in sound level at 256 and 1024 Hz modulation frequencies. The AMFR amplitudes were also not correlated with the ABR wave I or wave III amplitudes elicited for broadband click stimuli presented at the same sound level suggesting that sustained AMFR provide complementary information to phasic ABR responses. The FFT amplitudes varied significantly between young and aged animals for SAM stimuli in the presence of background noise, depending on the modulation frequency used and signal to noise ratio. The results show that the representation of temporally modulated stimuli is similar between young and aged animals in quiet listening conditions, but diverges substantially with the addition of background noise. This is consistent with a decrease in inhibition causing altered temporal processing with age

Environmental Modification of Bluegill (\u3cem\u3eLepomis macrochirus\u3c/em\u3e Rafinesque) in Chilhowee, Loudon, and Norris Reservoirs, Tennessee

This thesis reports the results of an investigation of environmental modifications of bluegill (Lepomis macrochirus Rafinesque) in Chilhowee, Loudon, and Norris Reservoirs. The three reservoirs are located in the Tennessee River drainage system. The investigation was carried out between the spring of 1968 and the fall of 1969. Collecting was done by angling using both natural and artificial bait. The evidence obtained by the investigation indicated significant differences occurred in the meristic and morphometric characters of the three bluegill populations when the data was tested statistically. It is concluded that the differences detected were probably due to environmental forces, but genetic differences may also have accumulated due to genetic drift. Even though highly significant differences were detected, no trends or patterns could be established linking the differences with the environmental forces causing the modifications. It is pointed out that further research into this question needs to be carried out by zoologists

Algebraic analysis of Trivium-like ciphers

Trivium is a bit-based stream cipher in the final portfolio of the eSTREAM project. In this paper, we apply the approach of Berbain et al. to Trivium-like ciphers and perform new algebraic analyses on them, namely Trivium and its reduced versions: Trivium-N, Bivium-A and Bivium-B. In doing so, we answer an open question in the literature. We demonstrate a new algebraic attack on Bivium-A. This attack requires less time and memory than previous techniques which use the F4 algorithm to recover Bivium-A's initial state. Though our attacks on Bivium-B, Trivium and Trivium-N are worse than exhaustive keysearch, the systems of equations which are constructed are smaller and less complex compared to previous algebraic analysis. Factors which can affect the complexity of our attack on Trivium-like ciphers are discussed in detail

The Determination of Carbonic Oxide in Illuminating Gas By Iodine Pentoxide

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A Computational Model of Cellular Mechanisms of Temporal Coding in the Medial Geniculate Body (MGB)

Acoustic stimuli are often represented in the early auditory pathway as patterns of neural activity synchronized to time-varying features. This phase-locking predominates until the level of the medial geniculate body (MGB), where previous studies have identified two main, largely segregated response types: Stimulus-synchronized responses faithfully preserve the temporal coding from its afferent inputs, and Non-synchronized responses, which are not phase locked to the inputs, represent changes in temporal modulation by a rate code. The cellular mechanisms underlying this transformation from phase-locked to rate code are not well understood. We use a computational model of a MGB thalamocortical neuron to test the hypothesis that these response classes arise from inferior colliculus (IC) excitatory afferents with divergent properties similar to those observed in brain slice studies. Large-conductance inputs exhibiting synaptic depression preserved input synchrony as short as 12.5 ms interclick intervals, while maintaining low firing rates and low-pass filtering responses. By contrast, small-conductance inputs with Mixed plasticity (depression of AMPA-receptor component and facilitation of NMDA-receptor component) desynchronized afferent inputs, generated a click-rate dependent increase in firing rate, and high-pass filtered the inputs. Synaptic inputs with facilitation often permitted band-pass synchrony along with band-pass rate tuning. These responses could be tuned by changes in membrane potential, strength of the NMDA component, and characteristics of synaptic plasticity. These results demonstrate how the same synchronized input spike trains from the inferior colliculus can be transformed into different representations of temporal modulation by divergent synaptic properties

Stalled flow performance of a single stage transonic compressor

Originally presented as the first author's thesis, M.S. in the M.I.T. Dept. of Mechanical Engineering, 1978Includes bibliographical references (page 48)The stalled flow performance of a single stage transonic compressor is examined, using data from the MIT Gas Turbine Laboratory's Blowdown Compressor Facility. Measurements of the blowdown corrected weightflow are included, as well as stage exit static to inlet total pressure rise, and rotating stall cell measurements. A comparison of the flow blockage, as represented by stall cell circumferential extent, with the blowdown corrected weightflow is made. The 100% design speed stalled flow performance characteristic is presented. Radial traverse.data is also presented including flow angles, static and total pressures, and Mach number components.Supported by the NASA Lewis Research Center grant NGL 22-009-38