Histones are proteins found in the nuclei of eukaryotic cells where they are complexed to DNA in chromatin. Rephcation-dependent histones are expressed only during S-phase. Regulation of expression of replication-dependent histone genes requires a highly conserved hairpin RNA element in the 3' untranslated region of histone mRNAs. Replication-dependent histone mRNAs are not polyadenylated; their 3' end is formed by an endonucleolytic cleavage event, 3' of a hairpin element, which is recognised by the Hairpin Binding Protein, HBP (also known as Stem-Loop Binding Protein, SLBP). This protein-RNA interaction is important for the endonucleolytic cleavage that generates the mature mRNA 3' end. The 3' hairpin, and presumably HBP, are also required for nucleocytoplasmic transport, translation and stability of histone mRNAs. It is therefore important to understand this interaction. The hairpin is highly conserved and I have demonstrated that residues in the hairpin loop are important for binding the HBP. This complimented structural studies that showed that the same residues are involved in stacking interactions in the RNA loop. In cell culture, expression of replication-dependent histone genes is S phase specific as is the expresion of HBP. Here I demonstrated that in Caenorhabditis elegans the HBP promoter is active in dividing cells during embryonic and postembryonic development. Depletion of HBP by RNAi leads to an embryonic lethal phenotype associated with defects in chromosome condensation. Postembryonic depletion of HBP results in defects in cell fate during late larval development, specifically in vulval development. A similar phenotype was obtained when histone H3 and H2A were depleted by RNAi suggesting that the phenotype of the hbp (RNAi) worms was due to a lack of histone proteins. I have confirmed this by showing that histone proteins are indeed reduced in hbp (RNAi) worms. I have also shown that depletion of HBP leads to a change in expression of a number of other proteins and specifically an up-regulation of a histone H3 like protein with an apparent molecular mass of 34 kDa. I have evidence that suggests that this protein is the centromer specific protein, CENP-A. As this protein was up-regulated when RNAi was used to deplete histones proteins, this suggests that there could be a compensatory mechanism that helps the animal to deal with the shortage of histone proteins.

Essa dissertação tem por objetivo o desenvolvimento de uma plataforma para testes de controladores de voo. Tal plataforma consiste em um hardware executando algoritmos de controle e atuando numa aeronave simulada em software de simulação de voo. O software de simulação escolhido, baseado na experiência prática de pilotos profissionais, foi o Microsoft Flight Simulator (MSFS), para o qual desenvolveu-se o modelo gráfico e dinâmico do quadricóptero AscTec Pelican. A comunicação entre o MSFS e o hardware é feita pela interface USB através do software FVMS v2.0 desenvolvido em ambiente DELPHI® 7.0 exclusivamente para este trabalho. O FVMS é capaz de ler o estado das variáveis de voo no MSFS, enviá-las para o hardware externo executar o controle, receber os sinais de controle de volta e utilizá-los no MSFS. O projeto e execução do hardware externo com controlador dsPIC também foi realizado neste mesmo trabalho. A título de avaliação de desempenho, também foi implementado um controlador robusto do tipo H∞ linear, desenvolvido pela equipe ART (Aerial Robots Team) da Escola de Engenharia de São Carlos. O mesmo controlador também foi aplicado na arquitetura software in the loop, na qual o controle é executado dentro do próprio FVMS, para comparação de desempenho entre os dois sistemas. Ao término do trabalho, as características de desempenho do sistema como um todo ficam bem evidenciadas através dos testes de estabilidade com e sem distúrbios executados em ambas arquiteturas de controle. / This dissertation aims to develop a platform for flight controllers tests. It platform consists of an electronic hardware where the control\'s algorithms will be executed and a virtual aircraft is simulated in flight simulation software. The chosen simulation software, based on practical experience of professional pilots, was Microsoft Flight Simulator (MSFS). The graphic and dynamic model of quadrotor AscTec Pelican was developed to perform inside the software. The communication between the MSFS and the hardware is made by USB interface through FVMS v2.0 software developed in DELPHI® 7.0 environment, exclusively for this work. The FVMS can read the status of the flight variables in MSFS, send them to the external hardware, receive control signals back and write them in MSFS. The design and implementation of external hardware with dsPIC controller was also developed ons ame work. For performance evaluation of the system, it was also implemented a robust linear H∞ controller, developed by ART team (Aerial Robots Team) of the School of Engineering of São Carlos. The same controller was also applied using software in the loop architecture, in which the control is performed inside FVMS, to compare performance between the two architectures. In the end of the work, the performance characteristics of the systems were well evidenced by the stability tests carried out with and without disturbances in both control architectures.

In a near future, it is imminent that passenger vehicles will soon be introduced with a new revolutionary brake by wire system which replaces all the mechanical linkages and the conventional hydraulic brake systems with complete 'dry' electrical components. One of the many potential benefits of a brake by wire system is the increased brake dynamic performances due to a more accurate and continuous operation of the EMB actuators which leads to the increased amount of possibilities for control in antilock brake system. The main focus of this thesis is on the application of a model predictive control (MPC) method to devise an antilock brake control system for a brake by wire vehicle. Unlike the traditional ABS control algorithms which are based on a trial and error method, the MPC based ABS algorithm aims to utilizes the behaviour of the model to optimize the wheel slip dynamics subject to system constraints. The final implementation of the wheel slip controller emb races decentralized control architecture to independently control the brake torque at each four wheel. Performance of the wheel slip controller is validated through Software-in-the-Loop and Hardware-in-the-Loop simulation. In order to support the high demands of the computational power and the real time constraints of the Hardware-in-the-Loop simulation, a novel multi processor real-time simulation system is developed using the reflective memory network and the off-the-shelf hardware components.

Flow speed describes general traffic operation conditions on a segment of roadway. It is also used to diagnose special conditions such as congestion and incidents. Accurate speed estimation plays a critical role in traffic management or traveler information systems. Data from loop detectors have been primary sources for traffic information, and single loop are the predominant loop detector type in many places. However, single loop detectors do not produce speed output. Therefore, speed estimation using single loop outputs has been an important issue for decades. This dissertation research presents two methodologies for speed estimation using single loop outputs. Based on findings from past studies and examinations in this research, it is verified that speed estimation is a nonlinear system under various traffic conditions. Thus, a methodology of using Unscented Kalman Filter (UKF) is first proposed for such a system. The UKF is a parametric filtering technique that is suitable for nonlinear problems. Through an Unscented Transformation (UT), the UKF is able to capture the posterior mean and covariance of a Gaussian random variable accurately for a nonlinear system without linearization. This research further shows that speed estimation is a nonlinear non-Gaussian system. However, Kalman filters including the UKF are established based on the Gaussian assumption. Thus, another nonlinear filtering technique for non-Gaussian systems, the Particle Filter (PF), is introduced. By combining the strengths of both the PF and the UKF, the second speed estimation methodology - Unscented Particle Filter (UPF) is proposed for speed estimation. The use of the UPF avoids the limitations of the UKF and the PF. Detector data are collected from multiple freeway locations and the microscopic traffic simulation program CORSIM. The developed methods are applied to the collected data for speed estimation. The results show that both proposed methods have high accuracies of speed estimation. Between the UKF and the UPF, the UPF has better performance but has higher computation cost. The improvement of speed estimation will benefit real-time traffic operations by improving the performance of applications such as travel time estimation using a series of single loops in the network, incident detection, and large truck volume estimation. Therefore, the work enables traffic analysts to use single loop outputs in a more cost-effective way.

Flow speed describes general traffic operation conditions on a segment of roadway. It is also used to diagnose special conditions such as congestion and incidents. Accurate speed estimation plays a critical role in traffic management or traveler information systems. Data from loop detectors have been primary sources for traffic information, and single loop are the predominant loop detector type in many places. However, single loop detectors do not produce speed output. Therefore, speed estimation using single loop outputs has been an important issue for decades. This dissertation research presents two methodologies for speed estimation using single loop outputs. Based on findings from past studies and examinations in this research, it is verified that speed estimation is a nonlinear system under various traffic conditions. Thus, a methodology of using Unscented Kalman Filter (UKF) is first proposed for such a system. The UKF is a parametric filtering technique that is suitable for nonlinear problems. Through an Unscented Transformation (UT), the UKF is able to capture the posterior mean and covariance of a Gaussian random variable accurately for a nonlinear system without linearization. This research further shows that speed estimation is a nonlinear non-Gaussian system. However, Kalman filters including the UKF are established based on the Gaussian assumption. Thus, another nonlinear filtering technique for non-Gaussian systems, the Particle Filter (PF), is introduced. By combining the strengths of both the PF and the UKF, the second speed estimation methodology—Unscented Particle Filter (UPF) is proposed for speed estimation. The use of the UPF avoids the limitations of the UKF and the PF. Detector data are collected from multiple freeway locations and the microscopic traffic simulation program CORSIM. The developed methods are applied to the collected data for speed estimation. The results show that both proposed methods have high accuracies of speed estimation. Between the UKF and the UPF, the UPF has better performance but has higher computation cost. The improvement of speed estimation will benefit real-time traffic operations by improving the performance of applications such as travel time estimation using a series of single loops in the network, incident detection, and large truck volume estimation. Therefore, the work enables traffic analysts to use single loop outputs in a more cost-effective way.

In this Masters dissertation, various doses of solubulized crude F4 fimbrial protein in conjunction with the adjuvants CpG ODN and porcine â-defensin 1 (pBD-1) were used to enhance the F4-specific intestinal immune response against Enterotoxigenic Escherichia coli (ETEC) F4 in post-weaned pigs. Using the mechanically shearing method we isolated the F4 fimbrial protein of ETEC with a molecular weight of 26 kDa. We verified this using a Western blot probed with a rabbit anti-F4 fimbrial antibody. Binding of the F4 fimbrial protein to the F4 receptor (F4R), present on the brush border of the villi in the small intestine of pigs, was demonstrated using an in vitro villus adhesion assay (IVVA). To demonstrate specificity rabbit polyclonal and mouse monoclonal anti-F4 antibodies, or the F4 protein were used to inhibit the adhesion of ETEC F4ac to F4R positive (F4Rpos) villi. <p>To examine immunogenicity of the 500 micrograms (ìg) of the F4 were administered into surgically created jejunal gut-loops in pigs. Three weeks later Peyers patches (PP) from immunized and control loops as well as gut-wall tissue were analyzed for their F4-specific antibody secreting cells (ASCs) by a modified enzyme linked immunosorbent spot (ELISPOT) assay. The F4-specific immune response in the serum was analyzed by an enzyme linked immunosorbant assay (ELISA). High numbers of F4-specific ASCs were isolated from the loops of pigs that contained high levels of the F4R. Conversely nominal or low numbers of F4-specific ASCs were found in loops of pigs expressing low levels of the F4R or no F4R (F4Rneg). The IVVA was used to categorize the pigs into either F4Rpos or F4Rneg animals. <p>Next three different concentrations of the crude F4 protein 50, 250, and 500 µg in the loops of individual pigs were used to analyze if dose affected the F4-specific immune response. Interestingly dose had no effect on the magnitude of the response. Therefore we hypothesized that the F4-specific immune response in the loops could be enhanced through the use of the adjuvants CpG ODN 2007 and pBD-1. The F4 protein was co-administered with either CpG ODN 2007 or pBD-1 and immune responses were assessed after 3 weeks. However neither CpG ODN 2007 nor PBD-1 at the doses used made an improvement in the immune response. Thus, these results demonstrated that the expression level of the F4R was the most important parameter for eliciting of the local immune response against the F4 protein. Furthermore our studies revealed that both F4Rneg and F4Rpos pigs responded to F4 immunization, however the former respond only nominally to F4-immunization in the loops. Moreover, an inverse relationship existed between the level of the F4-specific IgG in the serum and the F4-specific immune response seen in the loops. Thus our findings have important implications for oral vaccination using fimbrial based antigens (Ags) that utilize a receptor for their immunogenicity. Our results indicate that only animals with high levels of enterocyte F4R will have the ability to elicit high levels of protective F4-specific anti-fimbrial antibodies in their intestine after oral immunization. Therefore unless an effective adjuvant is available, animals with low to moderate levels of the fimbrial receptor in their small intestine will mount only weak immune responses making herd immunity after vaccination currently unattainable.

Organisms express metal-binding proteins in order to deal with essential metal ions that can be potentially toxic. A common trend of bacterial metal homeostasis pathways is the presence of a GTPase, and several of these proteins are members of the G3E family of P-loop GTPases. In this work we focused on E. coli YeiR, member of the under-characterized COG0523 subfamily, and on M. thermoacetica AcsF. The in vitro metal-binding properties of isolated YeiR are presented. The protein binds Ni2+ and Zn2+ with low micromolar affinity, and oligomerizes in the presence of metal. The GTPase activity of YeiR is similar to that measured for other members of the group and is enhanced by Zn2+. In the case of AcsF, it was not possible to establish concluding evidence that the protein binds metal. This study helps shed light upon members of the under-characterized subfamily of G3E P-loop GTPases.

Organisms express metal-binding proteins in order to deal with essential metal ions that can be potentially toxic. A common trend of bacterial metal homeostasis pathways is the presence of a GTPase, and several of these proteins are members of the G3E family of P-loop GTPases. In this work we focused on E. coli YeiR, member of the under-characterized COG0523 subfamily, and on M. thermoacetica AcsF. The in vitro metal-binding properties of isolated YeiR are presented. The protein binds Ni2+ and Zn2+ with low micromolar affinity, and oligomerizes in the presence of metal. The GTPase activity of YeiR is similar to that measured for other members of the group and is enhanced by Zn2+. In the case of AcsF, it was not possible to establish concluding evidence that the protein binds metal. This study helps shed light upon members of the under-characterized subfamily of G3E P-loop GTPases.

If the primary sequence of a protein is known, what is its three-dimensional structure? This is one of the most challenging problems in molecular biology and has many applications in proteomics. During the last three decades, this issue has been extensively researched. Techniques such as the protein folding approach have been demonstrated to be promising in predicting the core areas of proteins - α-helices and β-strands. However, loops that contain no regular units of secondary structure elements remain the most difficult regions for prediction. The protein loop prediction problem is to predict the spatial structure of a loop given the primary sequence of a protein and the spatial structures of all the other regions. There are two major approaches used to conduct loop prediction – the ab initio folding and database searching methods. The loop prediction accuracy is unsatisfactory because of the hypervariable property of the loops. The key contribution proposed by this thesis is a novel fragment assembly algorithm using branch-and-cut to tackle the loop prediction problem. We present various pruning rules to reduce the search space and to speed up the finding of good loop candidates. The algorithm has the advantages of the database-search approach and ensures that the predicted loops are physically reasonable. The algorithm also benefits from ab initio folding since it enumerates all the possible loops in the discrete approximation of the conformation space. We implemented the proposed algorithm as a protein loop prediction tool named LoopLocker. A test set from CASP6, the world wide protein structure prediction competition, was used to evaluate the performance of LoopLocker. Experimental results showed that LoopLocker is capable of predicting loops of 4, 8, 11-12, 13-15 residues with average RMSD errors of 0.452, 1.410, 1.741 and 1.895 A respectively. In the PDB, more than 90% loops are fewer than 15 residues. This concludes that our fragment assembly algorithm is successful in tackling the loop prediction problem.

If the primary sequence of a protein is known, what is its three-dimensional structure? This is one of the most challenging problems in molecular biology and has many applications in proteomics. During the last three decades, this issue has been extensively researched. Techniques such as the protein folding approach have been demonstrated to be promising in predicting the core areas of proteins - α-helices and β-strands. However, loops that contain no regular units of secondary structure elements remain the most difficult regions for prediction. The protein loop prediction problem is to predict the spatial structure of a loop given the primary sequence of a protein and the spatial structures of all the other regions. There are two major approaches used to conduct loop prediction – the ab initio folding and database searching methods. The loop prediction accuracy is unsatisfactory because of the hypervariable property of the loops. The key contribution proposed by this thesis is a novel fragment assembly algorithm using branch-and-cut to tackle the loop prediction problem. We present various pruning rules to reduce the search space and to speed up the finding of good loop candidates. The algorithm has the advantages of the database-search approach and ensures that the predicted loops are physically reasonable. The algorithm also benefits from ab initio folding since it enumerates all the possible loops in the discrete approximation of the conformation space. We implemented the proposed algorithm as a protein loop prediction tool named LoopLocker. A test set from CASP6, the world wide protein structure prediction competition, was used to evaluate the performance of LoopLocker. Experimental results showed that LoopLocker is capable of predicting loops of 4, 8, 11-12, 13-15 residues with average RMSD errors of 0.452, 1.410, 1.741 and 1.895 A respectively. In the PDB, more than 90% loops are fewer than 15 residues. This concludes that our fragment assembly algorithm is successful in tackling the loop prediction problem.