Originally 2A was characterised in foot-and-mouth disease virus. Site directed mutagenesis identified a C-terminus consensus motif [D(V/I)ExNPGP] and it is proposed that 2A interacts with the exit tunnel of the ribosome in a way that a specific peptide bond is skipped between the last glycine of 2A and the proline of 2B, thus providing a discontinuity in translation, resulting in release of discrete proteins from one single ORF. 2A was also identified in other picornaviruses, positive, single and double-stranded RNA insect viruses and mammalian rotaviruses. A motif present at the C-terminus of the 2A oligopeptide [D(V/I)ExNPGP] is very highly, though not completely conserved . The sequence upstream of this motif shows, however, no apparent conservation between 2As of different viruses. In this study, extensive site-directed mutagenesis were performed on several 2A sequences and a series of ‘hybrid' 2As comprising different consensus motifs juxtaposed with different upstream contexts were created as part of a detailed analysis of the mechanism of 2A-mediated ribosome stalling. The results demonstrated that a minimal region of twenty to twenty-three amino acids interacts with the exit tunnel of the ribosome to bring about a pause in processivity, alter the peptidyl transferase centre geometry and restrict the ribosome A site via two distinctive stalling mechanisms. Other molecular analyses tested here will require further optimisations or alternative methods: a visual method to explore the dynamics of re-initiation of translation from proline codon, purification of the translation-regulating factors and structural resolution of 2A sequences. Previously, cellular 2As were identified in non-LTR retrotransposons of trypanosomes. It is reported here as part of two other cellular organisms Saccoglossus kowalevskii (acorn worm) and Branchiostoma floridae (amphioxus). In the acorn worm, the nucleotides sequences corresponding to 2A motifs were part of the untranslated genome. In amphioxus, three 2A elements were identified in hypothetical proteins, and at the N-terminus of twenty non-LTR retrotransposons.

<p>Prema najnovijim istraživanjima modifikovanim skrobovima može se pripisati jo&scaron; jedna uloga &ndash; aditivi u pekarstvu, s obzirom da utiču na pobolj&scaron;anje kvaliteta hleba i<br />usporavaju proces starenja. Međutim, prema saznanjima autora ove disertacije, ne<br />postoje istraživanja na temu uticaja emulgujućih skrobova (skrob-natrijum<br />oktenilsukcinata, OSA skrobova) na kvalitet testa i hleba od p&scaron;eničnog bra&scaron;na.<br />Uzimajući u obzir činjenicu da druge vrste modifikovanih skrobova ispoljavaju dobre<br />osobine u smislu pobolj&scaron;anja kvaliteta hleba i da je OSA skrob nutritivno vredna<br />sirovina, s obzirom da se u organiznu pona&scaron;a kao prehrambeno vlakno, od velike je<br />važnosti ispitati ulogu OSA skroba kao aditiva u pekarstvu.<br />Međutim, usled kompleksne prirode testa kao sistema, bilo je te&scaron;ko odrediti relativni<br />uticaj pojedinačnih komponenti (proteina, p&scaron;eničnog skroba, OSA skroba) i njihovih<br />međusobnih interakcija na osobine testa. Stoga je, u cilju rasvetljavanja uticaja različitih OSA skrobova na viskoelastične osobine testa i retrogradaciju skrobne komponente (fenomen povezan sa starenjem hleba), p&scaron;enično bra&scaron;no frakcionisano na gluten i skrob, i vr&scaron;ena su ispitivanja strukturnih, reolo&scaron;kih i termičkih osobina hidratisanog glutena i skrobnih gelova sa dodatkom OSA skroba. Takođe su pripremani i model sistemi testa.<br />Ispitivanja na realnim sistemima (testo i hleb) izvođena su zamenom 2,5; 5 i 10%<br />p&scaron;eničnog bra&scaron;na, jednim od tri vrste OSA skroba: skrob-natrijum oktenilsukcinatom<br />(OSA-ST), preželatiniziranim skrob-natrijum oktenilsukcinatom (Pregel OSA-ST) i<br />hidrolizovanim su&scaron;enim u spreju skrob-natrijum oktenilsukcinatom (Hydrol OSA-ST).<br />Cilj je bio da se ispita uticaj dodatka OSA skrobova na strukturu testa (SEM), empirijske (miksolab, alveograf i reofermentometar) i fundamentalne (oscilatorna merenja i testovi puzanja i deformacije) reolo&scaron;ke osobine, kao i parametre kvaliteta hleba (specifična zapremina, boja kore i sredine hleba, vlaga sredine hleba, parametri raspodele veličine pora i tekstura sredine). Pored pomenutog, praćena je i promena kvaliteta hleba tokom skladi&scaron;tenja, kako bi se dobio uvid u kinetiku starenja hleba.<br />Rezultati dobijeni u ovoj disertaciji ukazali su da reolo&scaron;ko pona&scaron;anje testa sa dodatkom OSA skrobova zavisi od strukture skrobnih granula, tj. od stepena dezintegracije i depolimerizacije granule tokom procesa modifikacije. Stoga je dodatak OSA-ST doveo do ojačavanja glutenske mreže &scaron;to se odrazilo na porast modula elastičnosti i testa i glutena. Nasuprot tome, Pregel and Hydrol OSA-ST uticali su na kontinualnost glutenske mreže koja je u njihovom prisustvu bila poroznija, a time su i dobijena testa bila mek&scaron;a i rastegljivija. Istraživanja na realnim i model sistemima (p&scaron;enični skrob sa dodatkom OSA skroba) ukazala su da sva tri OSA skroba usporavaju retrogradaciju p&scaron;eničnog skroba. Takođe je dokazano da svi ispitivani emulgujući skrobovi povećavaju specifičnu zapreminu hleba u poređenju sa kontrolnim, pri čemu je Pregel OSA ispoljio najjači efekat. To se odrazilo i na strukturne osobine sredine hleba, a posledično i na mehaničke osobine sredine, čija promena je praćena u cilju određivanja kinetike starenja hleba. Prema dobijenim rezultatima, vrednosti čvrstoće sredine hleba koji je sadržao OSA-ST i Pregel OSA-ST su i nakon 24 h bile slične ili značajno niže od vrednosti čvrstoće kontrolnog uzorka određene 2 h nakon pečenja.</p> / <p>Recent studies have demonstrated that modified starches can be used as novel additives in breadmaking since they improve bread quality and retard stalling. However, up to the author&#39;s knowledge, there are no investigations concerning the influence of emulsifying starches (starch sodium octenyl succinates - OSA starches) on wheat flour dough and bread quality. Taking into account the fact that other modified starches have exhibited significant bread improving properties and that OSA starch has special nutritional value since it can act as functional fibre, it is of a great importance to investigate the feasibility of OSA starch as bread improver.<br />However, due to the complex nature of dough, it was difficult to determine the relative contributions of protein, native and modified starch components and their interactions on dough properties. Therefore, in order to resolve the influence of different OSA starches on dough viscoelastic properties and starch retrogradation (the phenomenon related to bread stalling), wheat flour was fractionated into gluten and starch; and the structural, rheological and thermal behaviour of the hydrated gluten samples and starch gels supplemented with emulsifying starches was also studied. Dough model systems were also prepared.<br />Experiments on real systems (dough and bread) were performed by incorporating<br />starch sodium octenyl succinate (OSA-ST), pregelatinized OSA starch (Pregel OSA-ST) and hydrolysed spray-dried OSA starch (Hydrol OSA-ST) at 2.5, 5 and 10 % into wheat flour. The aim was to investigate the effect of incorporating OSA starches on dough structural (SEM imaging), empirical (Mixolab, Alveograph, Rheofermentometre) and fundamental (oscillatory and creep measurements) rheological properties as well as bread quality parameters (specific loaf volume, crust and crumb colour, crumb moisture, crumb grain features, crumb texture). In addition, the bread quality attributes during storage were also monitored in order to get insight into bread stalling kinetics.<br />The results obtained in this thesis revealed that the rheological behaviour of OSA starch supplemented dough depended on the OSA starch granule rigidity, i.e. extent of OSA starch granule disintegration and polysaccharide degradation during modification. OSA-ST starch caused a reinforcement of the gluten network, as shown by the increase in storage modulus of doughs and gluten. On contrary, Pregel and Hydrol OSA-ST affected the continuity of gluten network which became porous and thus produced softer and stickier doughs in comparison to control. Investigations on real dough and model systems containing wheat and OSA starches revealed that all three types of OSA starches reduced starch retrogradation.<br />In general, all the examined emulsifying starches increased bread loaf volume in comparison to control bread with no added polymers, while Pregel OSA starch has expressed the greatest impact. It also reflected on bread crumb structural features and consequently on the crumb mechanical properties which were used for bread stalling monitoring. Firmness values of OSA-ST and Pregel OSA-ST starch supplemented bread crumbs, after 24 h of storage, were similar to or significantly lower than those of control determined 2 h after baking.</p>

Axial compressors are used in a wide variety of aerodynamic applications and are one of the most important components in aero-engines. The operability of compressors is however limited at low-mass flow rates by fluid dynamic instabilities such as stall and surge. These instabilities can lead to engine failure and loss of engine power which can compromise the aircraft safety and reliability. Therefore, a better understanding of how stall occurs and the causes behind its inception is extremely important. In the vicinity of the stall line, the flow field is inherently unsteady due to the interactions between adjacent rows of blades, formation of separation cells, and the viscous effects including shock-boundary layer interaction. Accurate modeling of these phenomena requires a proper set of stable and accurate boundary conditions at the rotorstator interface that conserve mass, momentum and energy, while eliminating false reflections. As a part of this effort, an existing 3D Navier-Stokes analysis for modeling single stage compressors has been modified to model multi-stage axial compressors and turbines. Several rotor-stator interface boundary conditions have been implemented. These have been evaluated for the first stage (a stator and a rotor) of the two stage fuel turbine on the space shuttle main engine (SSME). Their effectiveness in conserving global properties such as mass, momentum, and energy across the interface, while yielding good performance predictions has been evaluated. While all the methods gave satisfactory results, a characteristic based approach and an unsteady sliding mesh approach are found to work best. Accurate modeling of the formation of stall cells requires the use of advanced turbulence models. As a part of this effort, a new advanced turbulence model called Hybrid RANS/KES (HRKES) has been developed and implemented. This model solves Menter's k--SST model near walls and switches to a Kinetic Eddy Simulation (KES) model away from walls. The KES model solves directly for local turbulent kinetic energy and local turbulent length scales, alleviating the grid spacing dependency of the length scales found in other Detached Eddy Simulation (DES) and Hybrid RANS/LES (HRLES) models. Within the HRKES model, combinations of two different blending functions have been evaluated for blending the near wall model to the KES model. The use of realizability constraints to bound the KES model parameters has also been studied for several internal and external flows. The current methodology is used in the prediction of the performance map for the NASA Stage 35 compressor configuration as a representative of a modern compressor stage. The present approach is found to satisfactory predict the onset of stall. It is found that the rotor blade tip leakage vortex and its interaction with the shock wave is mainly the reason behind the stall inception in this compressor stage.

Studies reported in this thesis address the question of how pre-termination ribosomal complexes stalled during translation of mRNA are recycled. The process of recycling of the stalled ribosomes involves many translational factors. During the course of my studies, I have uncovered new roles of SsrA (tmRNA), IF3 and ribosome recycling factor (RRF) in recycling stalled ribosomes. These findings are summarized as follows: (i) A physiological connection between tmRNA and peptidyl-tRNA hydrolase functions in Escherichia coli The bacterial ssrA gene codes for a dual function RNA, tmRNA, which possesses tRNA-like and mRNA-like regions. The tmRNA appends an oligopeptide tag to the polypeptide on the P-site tRNA by a trans-translation process that rescues ribosomes stalled on mRNAs and targets the aberrant protein for degradation. In cells, processing of the stalled ribosomes is also pioneered by drop-off of peptidyl-tRNAs. The ester bond linking the peptide to tRNA is hydrolyzed by peptidyl-tRNA hydrolase (Pth), an essential enzyme, which releases the tRNA and the aberrant peptide. As the trans-translation mechanism utilizes the peptidyl-transferase activity of the stalled ribosomes to free the tRNA (as opposed to peptidyl-tRNA drop-off), the need for Pth to recycle such tRNAs is bypassed. Thus, we hypothesized that tmRNA may rescue a defect in Pth. The findings of the experiments detailed in this thesis show that SsrA rescues a defect in Pth by reducing the peptidyl-tRNA load on Pth. (ii) Evidence for a role of initiation factor 3 in recycling ribosomal complexes stalled on mRNAs in Escherichia coli. Specific interactions between ribosome recycling factor (RRF) and EF-G mediate disassembly of post-termination ribosomal complexes for new rounds of initiation. The interactions between RRF and EF-G are also important in peptidyl-tRNA release from pre-termination complexes. Unlike the post-termination complexes (harboring tRNA), the pre-termination complexes (harboring peptidyl-tRNA) are not recycled by RRF and EF-G in vitro, suggesting participation of additional factor(s) in the process. Using a combination of biochemical and genetic approaches, we show that, 1. Inclusion of IF3 with RRF and EF-G results in recycling of the pre-termination complexes; 2. IF3 overexpression in Escherichia coli LJ14 rescues its temperature sensitive phenotype for RRF; (3) Transduction of infC135 (encoding functionally compromised IF3) in E. coli LJ14 generates a ‘synthetic severe’ phenotype; (4) The infC135 and frr1 (a promoter down RRF gene) alleles synergistically rescue a temperature sensitive mutation in peptidyl-tRNA hydrolase in E. coli; and (5) IF3 facilitates ribosome recycling by Thermus thermophilus RRF and E. coli EFG in vivo and in vitro. These lines of evidence clearly demonstrate the physiological importance of IF3 in the overall mechanism of ribosome recycling in E. coli. (iii) The role of RRF in dissociating of pre-termination ribosomal complexes stalled during elongation Translating ribosomes often stall during the repetitive steps of elongation for various reasons. The stalled ribosomes are rescued by the process of trans-translation involving tmRNA (SsrA) or by a factor mediated dissociation of the stalled ribosome into its subunits leading to the drop-off of the peptidyl-tRNA. The mechanistic details of how the factor mediated dissociation is carried out, is not well studied. Studies described in the above section have highlighted the role of RRF in dissociating stalled pre-termination complexes. However, the in vivo studies in this area have been limited for lack of defined pre-termination complexes. Two in vivo systems based on translation of AGA minigene and the ung gene (EcoUngstopless) transcripts were designed. Evidence is presented to show that translation of both of these transcripts is toxic to E. coli because of the accumulation of the transcript specific stalled pre-termination complexes. Availability of these model systems has allowed us to address the role of RRF in dissociating stalled ribosomes. We show that RRF rescues stalled ribosomes on these constructs and its overexpression can rescue the toxicity. The physiological importance of this observation is highlighted by the rescue of AGA minigene inhibitory effect on λimmP22 hybrid phage growth upon RRF overexpression.

In response to DNA damage during S phase, cells slow DNA replication. This slowing is orchestrated by the intra-S checkpoint and involves inhibition of origin firing and reduction of replication fork speed. Slowing of replication allows for tolerance of DNA damage and suppresses genomic instability. Although the mechanisms of origin inhibition by the intra-S checkpoint are understood, major questions remain about how the checkpoint regulates replication forks: Does the checkpoint regulate the rate of fork progression? Does the checkpoint affect all forks, or only those encountering damage? Does the checkpoint facilitate the replication of polymerase-blocking lesions? To address these questions, we have analyzed the checkpoint in the fission yeast Schizosaccharomyces pombe using a single-molecule DNA combing assay, which allows us to unambiguously separate the contribution of origin and fork regulation towards replication slowing, and allows us to investigate the behavior of individual forks. Moreover, we have interrogated the role of forks interacting with individual sites of damage by using three damaging agents—MMS, 4NQO and bleomycin—that cause similar levels of replication slowing with very different frequency of DNA lesions. We find that the checkpoint slows replication by inhibiting origin firing, but not by decreasing fork rates. However, the checkpoint appears to facilitate replication of damaged templates, allowing forks to more quickly pass lesions. Finally, using a novel analytic approach, we rigorously identify fork stalling events in our combing data and show that they play a previously unappreciated role in shaping replication kinetics in response to DNA damage.

No description available.

Purpose - The paper presents a theoretical framework that describes the aerodynamics of a falling maple (Acer pseudoplatanus) seed. --- Methodology - A semi-empirical method is developed that provides a ratio stating how much longer a seed falls in air compared to freefall. The generated lift is calculated by evaluating the integral of two-dimensional airfoil elements using a preliminary falling speed. This allows for the calculation of the definitive falling speed using Blade Element Momentum Theory (BEMT); hereafter, the fall duration in air and in freefall are obtained. Furthermore, the input-variables of the calculation of lift are transformed to require only the length and width of the maple seed. Lastly, the method is applied to two calculation examples as a means of validation. --- Findings - The two example calculations gave percentual errors of 5.5% and 3.7% for the falling speed when compared to measured values. The averaged result is that a maple seed falls 9.9 times longer in air when released from 20 m; however, this result is highly dependent on geometrical parameters which can be accounted for using the constructed method. --- Research limitations - Firstly, the coefficient of lift is unknown for the shape of a maple seed. Secondly, the approximated transient state is yet to be verified by measurement. --- Originality / Value - The added value of this report lies in the reduction of simplifications compared to BEMT approaches. In this way a large amount of accuracy is achieved due to the inclusion of many geometrical parameters, even though simplicity is maintained. This has been accomplished through constructing a simple three-step method that is fundamental and essentially non-iterative.

The aim of this thesis is to break new ground by investigating the interactional organisation of real events that comprise live business-to-business cold calls. Despite being a ubiquitous part of everyday life, we know very little about how cold calls are initiated, progressed, and completed. Cold calls are unsolicited telephone encounters, initiated by salespeople aiming to get prospective clients ( prospects ) interested in their services, with the distal goal of turning them into clients and the proximal goal of getting them to agree to an initial meeting. Cold calls are often treated as a nuisance by call-takers, and salespeople must deal with reluctant gatekeepers, recurrent sales resistance, and the occasional hang-up. The training they receive often draws on outdated theories of communication and is rarely supported by empirical evidence. Thus, this study not only addresses an important domain for interactional research, but also fulfils a practical necessity for empirical research that will inform sales training and improve callers and call-takers experiences. The data comprise 150 recorded calls supplied by three British companies that sell, service, and lease office equipment. The data were collected, transcribed, and analysed within an ethnomethodological framework using conversation analysis and discursive psychology. The first analytic chapter outlines the overall structural organisation of cold calling. It documents the constituent activities within the opening, the business of the call, and the closing. It identifies and describes two types of cold calls. Freezing calls are initiated by salespeople who are contacting a prospect for the very first time. Lukewarm calls feature salespeople who claim to have been in contact with the prospect s organisation in the past. The second chapter excavates the initial turns of lukewarm calls in which salespeople ask to speak to another person within the company, with whom they indicate to be acquainted. The analysis revealed that this third-party acquaintanceship was crucial for establishing the legitimacy of the switchboard request and for improving the chances of getting it granted. The third chapter focused on appointment-making sequences in both freezing and lukewarm calls, showing that they comprise two components: a preamble and a meeting request sequence. I also highlight how salespeople exploit sequential and turn-taking mechanisms to secure meetings with prospects without giving the latter the opportunity to refuse. The final chapter examines two practices for enacting resistance in cold calls blocks and stalls and documents the range of methods salespeople employ for dealing with each type of resistance. Sales blocks expose the salesperson s commercial agenda, attempt to stop the prospecting activity, and move towards call pre-closure. In response, salespeople can challenge, counter, or circumvent blocks as well as redo their initiating actions. Stalls slow down the progress of the sales process by delaying the next phase of the sale or by proposing less commitment-implicative alternatives. Salespeople deal with stalls by either justifying their initial proposal or by spontaneously introducing new action plans, both being more conducive to the progress of the sale. The thesis contributes to a growing body of interactional research on commercial encounters by shedding empirical light on a previously unexamined setting, business-to-business cold calls. It also moves forward discursive psychology s project of respecifying psychological phenomena by documenting the communicative practices associated with persuasion and resistance. Finally, it expands the extant conversation analytic toolkit by examining new practices (such as appointment-making) and by providing new insights into key conversation analytic topics (such as requests, pre-sequences, and accounts for calling). Overall, the findings presented in this thesis challenge existing conceptions of prospecting through cold calling that are prevalent in the sales literature. The thesis puts forward a strong argument for opening the black box of cold calls to better understand these interactions and to identify good practices as the basis for communication training. Research presented in this thesis has already been used in the development of CARM (Conversation Analytic Role-play Method) training for salespeople, who reported having doubled their appointment rates. Based on the findings in this thesis, I plan to develop further training not only for salespeople but also for prospective customers, thus improving the overall outcome of cold call encounters.