Proteínas Inativadoras de Ribossomos (RIPs) são rRNA N-glicosilases capazes de inibir a síntese protéica pela remoção de uma adenina específica do RNA ribossomal. São geralmente classificadas em tipo 1 e tipo 2, sendo as últimas divididas em altamente tóxicas e não tóxicas. A maior parte das RIPs tipo 2 identificadas pertence a espécies de dicotiledôneas, como é o caso da pulchellina. As cadeias tóxicas das RIPs possuem uma região C-terminal hidrofóbica conservada, a qual se atribui a capacidade de interação com a membrana do retículo endoplasmático (RE), durante o transporte retrógrado da toxina para o citosol. Neste trabalho duas abordagens diferentes foram aplicadas para o estudo das RIPs tipo 2: identificação e caracterização de novos integrantes desta família de proteínas, e investigação da interação da cadeia-A da pulchellina (PAC) com sistemas miméticos da membrana celular. Na primeira abordagem, uma busca in silico em bancos de dados genéticos públicos permitiu identificar quatro novas RIPs do tipo 2 de monocotiledôneas. A análise da estrutura primária das proteínas identificadas mostrou a ocorrência de mutações em alguns dos principais aminoácidos que formam o sítio ativo nas RIPs, indicando uma possível perda de função. O representante de Saccharum officinarum (cana-de-açúcar) foi então analisado em maior detalhe, sendo sua cadeia-A clonada (soRIPA), expressa em sistema heterólogo e caracterizada em termos de atividade e estrutura secundária. Os ensaios in vitro mostraram que a soRIPA não foi capaz de depurinar ribossomos eucariotos. Porém, os ensaios de inibição da síntese proteica mostraram uma possível atividade inibitória da soRIP, que precisa ainda ser confirmada. A presença dos transcritos no banco do SUCEST sugere que estes genes não sejam pseudogenes, embora não tenha sido possível purificar a proteína a partir de extratos de folhas. Isto indica que se a soRIP está sendo traduzida, deve sofrer um rápido turnover, tornando difícil a sua detecção e purificação ou, ainda, que a ausência de sítios de ligação à galactose funcionais na cadeia-B impediu sua purificação por cromatografia de afinidade à galactose. A outra abordagem no estudo das RIPs tipo 2 foi centrada na cadeia-A recombinante da pulchellina (rPAC), estudando sua interação com monocamadas de Langmuir. Foram construídos 3 mutantes da rPAC, cada um com diferentes deleções na região C-terminal visando determinar a região responsável pela interação com a membrana do RE. A cinética de adsorção e pressão superficial exercida pela rPAC sobre a monocamada, assim como o estudo com os mutantes demontraram que a proteína interage fortemente com a monocamada fosfolipídica e que esta interação in vitro é dependente da presença da região C-terminal. De forma geral, os resultados obtidos neste trabalho contribuíram com novas informações sobre esta família de proteínas, identificando e analisando novos integrantes e, ainda, adicionando detalhes do mecanismo funcional do tráfego das toxinas RIPs. / Ribosome Inactivating Proteins (RIPs) are rRNA N-glycosilases which are able to inhibit the protein synthesis by removing a specific adenine from the ribosomal RNA. They are usually classified as type 1 and type 2, being the latter divided into highly toxic and nontoxic. The majority of type 2 RIPs currently identified are found in species of dicotyledons, as the pulchellin. The toxic chain of RIPs has a conserved hydrophobic C-terminal region, which is believed to be responsible for the interaction with the lipid membrane of the endoplasmic reticulum ER during the retrograde transport of the toxin to the cytosol. In this work, two different approaches were applied in the study of type 2 RIPs: identification and characterization of new members of this protein family, and investigation of the interaction of the pulchellin\'s A-chain (PAC) with systems that mimic the cellular membrane. In the first approach, an in silico search in public genetic databases was performed and allowed us to identify four new type 2 RIPs in monocots. The primary structure analysis of the identified proteins showed the presence of mutations in key amino acids that form the active site of RIPs, indicating a possible interference on its catalytic activity. The representative of Saccharum officinarum (sugar cane) was then analyzed in greater detail. Its A-chain clone (soRIPA) was expressed in a heterologous system and characterized in terms of activity and secondary structure. In vitro experiments showed that soRIPA was not able to perform the depurination of eukaryotic ribosomes. However, the inhibition of protein synthesis assays presented a possible low inhibitory activity of the soRIP, which still needs to be further investigated. The presence of transcripts on the bank of SUCEST indicates that these genes are not pseudogenes, although it was not possible to purify the protein from leaf extracts. If the soRIP is being translated, this may indicate that it undergoes a quick turnover, preventing its detection and purification. It is also possible that the absence of functional galactose binding sites in the B-chain has prevented its purification by galactose affinity chromatography. Our second approach to the study of type 2 RIPs was focused on the recombinant pulchellin A-chain (rPAC), by investigating its interaction with Langmuir monolayers. We have constructed three mutants of rPAC, each one with different deletions at the C-terminal to determine the region responsible for interaction with the membrane of the ER. The adsorption kinetic and surface pressure applied by rPAC on the monolayer, as well as the study of the mutants, have demonstrated that the protein has a strong interaction with the phospholipid monolayer and that this interaction in vitro is dependent on the presence of the C-terminal. The results of this work have provided new information about the type 2 RIP protein family, identifying and analyzing new members, and also bringing new details about the functional mechanism of the RIP\'s toxin traffic.

Hurricanes are one of the primary threats to the Texas coastal environment and economy. They generate large wave and storm surges that have caused much damage on the Texas coast in the past. Understanding both the hydrodynamic processes that damage coastal habitats and hurricane hazard and risk are critical to preserve coastal vegetation and quantify its benefits to coastal storm protection. The goal of this project is to quantify the impact of wave attenuation and wave refraction as well as the development of coherent structures in marsh fringes and the formation of a rip current system over wetlands on storm protection. The 3D Shallow Water Wave Basin at Texas A&M University hosted a series of large-scale experiments considering an idealized wetland model to pursue this goal. Study of the marsh geometry of the Texas coast was done in order to scale the experiments to the size of the Haynes Laboratory 3D-Water Wave Basin using a Froude and a Reynolds scalings. Particularly, averaged size and idealized shape of marsh segments in the area of Dalehite Cove in Galveston Bay were considered. Three sets of different wave conditions and water levels were tested to approximate different intensities of storm surge. Identical tests with both vegetated and non-vegetated marshes were run to compare the influence of the vegetation in storm conditions, and three different spacings between marsh segments were tested. In the basin, normally incident regular waves were generated at three water circulation structures. Data analysis allows the determination of the impact of discontinuous marsh segments on wave attenuation and wave refraction. Coherent structures such as rip current and the circulation pattern were analyzed to study the change in the flow field during passage of the waves. The experimental measurements were able to describe the wave transformations over the marsh segments. The influence of coastal wetlands was identified to affect the hydrodynamic process and reduce the total wave energy which is dissipated and redistributed by vegetation. The presence of the mounds induced an important decrease in the wave height, in addition to the damping of the waves by the vegetation stems. The variation in spatial coverage of the wetland model has been shown to highly affect the flow dynamics by generating offshore directed flow in the channel and onshore directed flow on the marsh mounds. This experimental approach provides a further understanding of flow dynamics by waves and surge in wetlands, at a large scale.

Magnetic Resonance Imaging (MRI) is now increasingly being used for fast imaging applications such as real-time cardiac imaging, functional brain imaging, contrast enhanced MRI, etc. Imaging speed in MRI is mainly limited by different imaging parameters selected by the pulse sequences, the subject being imaged and the RF hardware system in operation. New pulse sequences have been developed in order to decrease the imaging time by a faster k-space scan. However, they may not be fast enough to facilitate imaging in real time. Parallel MRI (pMRI), a technique initially used for improving image SNR, has emerged as an effective complementary approach to reduce image scan-time. Five methods, viz., SENSE [Pruesmann, 1999], PILS [Griswold, 2000], SMASH [Sodickson, 1997], GRAPPA [Griswold, 2002] and SPACE RIP [Kyriakos, 2000]; developed in the past decade have been studied, simulated and compared in this research. Because of the dependence of the parallel imaging methods on numerous factors such as receiver coil configuration, k-space subsampling factor, k-space coverage in the imaging environment, there is a critical need to find the method giving the best results under certain imaging conditions. The tools developed in this research help the selection of the optimal method for parallel imaging depending on a particular imaging environment and scanning parameters. Simulations on real MR phased-array data show that SENSE and GRAPPA provide better image reconstructions when compared to the remaining techniques.

The research presented in this dissertation focuses on understanding the dynamics of waves and currents in the presence of wave breaking. The simplest approach, direct numerical simulation of the ocean dynamics, is computationally prohibitive--waves typically have periods of tens of seconds, while currents vary on times from hours to days. This work uses a multi-scale asymptotic theory for the waves and currents (Craik and Leibovich, 1976; McWilliams et al., 2004}, similar to Reynolds-averaged Navier-Stokes, in order to avoid resolving the wave field. The theory decomposes the total flow into the mean flow (current) and fluctuations (waves), then takes a moving time average of the total flow equations to determine the wave forcing on the current. The main challenge is extending this theory to include a physical model for dissipative wave effects, notably breaking, in terms of the wave age, wind speed, and bottom topography. Wave breaking is difficult to observe, model, and predict, because it is an unsteady, non-linear process that takes place over disparate scales in both space and time. In the open ocean, white-capping often covers less than 2% of the surface, yet still plays an important role in the flux of mass, momentum, heat, and chemicals between the atmosphere and ocean. The first part of this dissertation proposes a stochastic model for white-capping events, and examines the stability of the ensemble average of these events. Near the shore, the decreasing ocean depth causes waves to overturn and break. Over time, this drives currents that erode sediment from beaches and deposit it around coastal structures. These currents are often so strong that their effect on the wave field, and thus their own forcing, is significant. A detailed analysis of this phenomena makes up the second part of this dissertation.

Rip currents present a severe hazard to water users worldwide, resulting in over 100 drownings and thousands of lifeguard rescues annually. This thesis examines the demographics of who is effected by rip currents in the UK, what activity they are undertaking, when and where incidents occur, how much the public know, what people have experienced, and how best to educate them. Analysis of 7909 rip current lifeguard rescues (16777 people) across the UK between 2006-2013 highlighted the most at risk group, and subsequent target audience for education, to be male teenagers aged between 13-17 years old (n=2906, 17%). Geographically, the highest incidents occurred on the beaches in the Southwest of England (n=6911, 87%). Incidents mostly occurred outside of lifeguard flagged areas (n=4302, 54%) and mainly involved those using bodyboards (n=5290, 52%). Through the analysis of 407 public beach-based rip current and beach safety questionnaires, it was established that beach users have a poor understanding of rip currents (n=263, 65%) but a good perception of the beach safety flags (n=389, 96%). People with greater knowledge were typically educated by a lifeguard, enter the sea more frequently or have been caught in rip currents themselves. The experiences of 553 people caught in rip currents were analysed using an online questionnaire. The gender split was 69% male (n= 382) to 31% female (n= 171), indicating that males are caught in rip currents more than females. Swimming directly to shore against the rip followed an initial panic (n=108, 34%) for most people caught in a rip. Swimming parallel to the beach was the most remembered, advised, utilised, and promoted safety message. Respondents advocated the use of lifeguards to disseminate rip current safety messages. A new and unique rip current education programme was developed from the synthesis of these results. A lifeguard delivered a pilot programme to 185 teenagers in three schools and two community groups in the Southwest of England. This interactive pilot consisted of exercises using videos, photographs, news reports, and a swimming machine. Levels of rip current knowledge were evaluated before and after, and at regular intervals, to assess knowledge retention. The short-term effects after 3 months were positive, showing statistically significant (p < 0.0005) improvements in mean knowledge levels. This thesis provides a new contribution to the expanding field of social and behavioural rip current research. The development of a unique rip current education programme presents an alternative method for increasing public awareness, and supports the worldwide prevention of rip current incidents and fatalities.

Le complexe EJC (Exon Junction Complex) joue un rôle central dans le couplage des processus post-transcriptionnels chez les métazoaires. Ce complexe multi-protéique est assemblé sur les ARN messagers (ARNm) par la machinerie d’épissage. Organisé autour d’un complexe cœur servant de plate-forme à de nombreux facteurs, les EJCs accompagnent les ARNm dans le cytoplasme et participent à leur transport, leur traduction et leur stabilité. L’importance physiologique de l’EJC est supportée par les nombreux défauts développementaux et les maladies génétiques associées aux composants de l’EJC. Les analyses transcriptomiques révélant un assemblage hétérogène des EJCs renforcent l’hypothèse que les EJCs participent à la régulation de l’expression des gènes. Cependant, malgré une connaissance précise de la structure de ce complexe, les liens fonctionnels entre l’assemblage de l’EJC et la régulation de transcrits spécifiques dans des conditions physiologiques doivent être établis puis caractérisés.Durant cette thèse, j’ai étudié l’expression d’eIF4A3, Y14 et MLN51, trois protéines du cœur de l’EJC, dans des cultures primaires de cellules souches neurales murines (CSN). Les CSN peuvent être différenciées en cellules épendymaires multi-ciliées qui tapissent les ventricules cérébraux et ont un rôle important dans le développement du cerveau. J’ai observé par immunofluorescence dans des CSN quiescentes que les 3 protéines sont concentrées autour du centrosome à la base du cil primaire. Ces localisations reflètent la présence d’EJC assemblés comme le prouve l’étude d’un mutant d’Y14 incapable de former l’EJC. / The Exon Junction Complex (EJC) plays a central role in coupling post-transcriptional processes in metazoans. This multi-protein complex is assembled onto messengers RNAs (mRNAs) by the splicing machinery. Organized around a core complex serving as a platform for numerous factors, EJCs accompany mRNAs to the cytoplasm and is involved in mRNA transport, translation and stability. The physiological importance of the EJC is supported by observations associating defects in EJC component expression to developmental defects and human genetic disorders. Transcriptomic studies revealing the non-ubiquitous deposition of EJCs strengthened the hypothesis that EJCs could participate to gene expression regulation. However, despite a precise picture of the structure of the EJC, functional links between EJC assembly and regulation of specific transcripts under physiological conditions is yet to be established.During this thesis, I studied the expression of eIF4A3, Y14 and MLN51 three core proteins of the EJC in primary cultures of mouse neural stem cells (NSCs). NSCs can be differentiated into multiciliated ependymal cells that line all brain ventricles and have important physiological functions in brain development. We observed by immunofluorescence that in quiescent NSCs, all three proteins are concentrated in the vicinity of the centrosome at the base of the primary cilia. This localization reflects the presence of fully assembled EJCs as proved by the study of Y14 mutant that prevent EJC core mounting.

京都大学 / 0048 / 新制・論文博士 / 博士(生命科学) / 乙第13281号 / 論生博第19号 / 新制||生||55(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 井垣 達吏, 教授 垣塚 彰, 教授 藤田 尚志 / 学位規則第4条第2項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

The aim of this Thesis is giving information about the network simulating system OMNeT++ and its extension INET framework and explore possible solution to implement missing protocol RIP. OMNeT++ is a discrete-event simulation environment. Source code is available and free for teaching and research purposes. Its primary application area is the simulation of communication networks, but because of its generic and flexible architecture, is successfully used in other areas like the simulation of complex IT systems, queueing networks or hardware architectures as well. The first part of Thesis decribes RIP protocol operations and conception of OMNeT++. At the second part of Thesis the implementation design and discussion of created modules and component is presented here. The final part of the Thesis was creating the laboratory problem on the basis experience gained. Implementation is realized in C++ programming language.

The aim of this thesis was to get acquainted with the technology of routing data in a computer network. Thesis described in the theoretical level properties of current and past routing protocols and their use in information systems. Based on the acquired knowledge was created a simulation model of network communication in programming environment OPNET Modeler. This model analyzes the received parameters of the current protocols and their subsequent implementation in the real transmission technology using operating system Debian Linux. Finally, the transfer parameters are discussed between the simulation model and real transfer technology.

A coarse-grain parallelization of SHORECIRC - a quasi-3D nearshore circulation model is implemented. The parallelization is based on the message-passing model for distributed memory architectures using the Message Passing Interface (MPI) standard. The parallel model confirms to the Single Program Multiple Data (SPMD) model. The results from the parallel model have been verified against those from the sequential model for an exact match. The parallel code is portable across different parallel architectures and its performance in terms of speed-up and scalability is studied. A test case simulating rip currents is discussed.