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Aedes aegypti transmits the viruses which cause yellow fever, dengue fever, and dengue hemorrhagic fever. Homing endonucleases are selfish genetic elements which introduce double-stranded DNA (dsDNA) breaks in a sequence-specific manner. In this study, we aimed to validate a somatic assay to detect recombinant homing endonuclease (rHE)-induced dsDNA breaks in both cultured cells and adult female Ae. aegypti. While the cell culture-based two plasmid assay used to test rHE ability to induce dsDNA breaks was inconclusive, assays used to test rHEs in Ae. aegypti were successful. Recognition sequences for various rHEs were introduced into Ae. aegypti through germline transformation, and imperfect repair at each of these exogenous sites was evaluated. In mosquitoes containing a single exogenous HE site, imperfect gap repair was detected in 40% and 21% of clones sequenced from mosquitoes exposed to I-PpoI and Iâ SceI, respectively. In mosquitoes containing two exogenous HE sites flanking a marker gene (EGFP), 100% of clones sequenced from mosquitoes exposed to I-PpoI, I-CreI, and I-AniI demonstrated excision of EGFP. No evidence of EGFP excision or imperfect repair at any HE recognition site was detected in mosquitoes not exposed to a rHE. In summary, a somatic genomic footprint assay was developed and validated to detect rHE or other meganuclease-induced site-specific dsDNA breaks in chromosomal DNA in Ae. aegypti. / Master of Science in Life Sciences

DYNAMIC MODELING, GUIDANCE, AND CONTROL OF HOMING MISSILES &Ouml / ZKAN, B&uuml / lent Ph. D., Department of Mechanical Engineering Supervisor: Prof. Dr. M. Kemal &Ouml / ZG&Ouml / REN Co-Supervisor: Dr. G&ouml / kmen MAHMUTYAZICIOgLU September 2005, 236 pages In this study, the dynamic modeling, guidance, and control of a missile with two relatively rotating parts are dealt with. The two parts of the missile are connected to each other by means of a roller bearing. In the first part of the study, the governing differential equations of motion of the mentioned missile are derived. Then, regarding the relative rotation between the bodies, the aerodynamic model of the missile is constructed by means of the Missile Datcom software available in T&Uuml / BiTAK-SAGE. After obtaining the required aerodynamic stability derivatives using the generated aerodynamic data, the necessary transfer functions are determined based on the equations of motion of the missile. Next, the guidance laws that are considered in this study are formulated. Here, the Linear Homing Guidance and the Parabolic Homing Guidance laws are introduced as alternatives to the Proportional Navigation Guidance law. On this occasion, the spatial derivation of the Proportional Navigation Guidance law is also done. Afterwards, the roll, pitch and yaw autopilots are designed using the determined transfer functions. As the roll autopilot is constructed to regulate the roll angle of the front body of the missile which is the controlled part, the pitch and yaw autopilots are designed to realize the command signals generated by the guidance laws. The guidance commands are in the form of either the lateral acceleration components or the flight path angles of the missile. Then, the target kinematics is modeled for a typical surface target. As a complementary part of the work, the design of a target state estimator is made as a first order fading memory filter. Finally, the entire guidance and control system is built by integrating all the models mentioned above. Using the entire system model, the computer simulations are carried out using the Matlab-Simulink software and the proposed guidance laws are compared with the Proportional Navigation Guidance law. The comparison is repeated for a selected single-body missile as well. Consequently, the simulation results are discussed and the study is evaluated.

O estudo da diversidade viral em amostras ambientais tem se tornado cada vez mais importante devido a funções-chave desempenhadas por esses organismos. Estudos recentes têm fornecido evidências de que vírus de bactérias (bacteriófagos) podem ser os principais determinantes em ciclos biogeoquímicos de grandes ecossistemas, além de atuarem no fluxo de genes entre comunidades ambientais e na plasticidade funcional das mesmas frente a estresses ambientais. Neste trabalho, propomos a investigação e caracterização da diversidade viral presente em amostras de compostagem através de abordagens não dependentes e dependentes de cultivo. Na primeira abordagem, coletamos amostras seriadas de uma unidade de compostagem do zoológico de São Paulo para realização de sequenciamento metagenômico. O conjunto de sequências gerado foi extensivamente minerado (data-mining) para a produção de resultados de diversidade e abundância de táxons virais ao longo do processo de compostagem. Adicionalmente, procedemos com a montagem e recuperação de sequências virais candidatas a genomas completos e/ou parciais de novos vírus ambientais. Os dois protocolos computacionais utilizados para a mineração de dados encontram-se definidos e automatizados, podendo ser aplicados em quaisquer conjuntos de dados de sequenciamento metagenômico ou metatranscritômico obtidos através da plataforma Illumina. A segunda abordagem correspondeu ao isolamento e caracterização de novos fagos de Pseudomonas obtidos de amostras de compostagem. Três novos fagos foram identificados e tiveram os seus genomas sequenciados. A caracterização genômica desses fagos revelou genomas com alto grau de novidade, insights sobre a evolução de Caudovirales e a presença de genes de tRNA, cuja função pode estar relacionada com um mecanismo dos fagos para contornar o viés traducional apresentado pela bactéria hospedeira. A caracterização experimental dos novos fagos isolados demonstrou grande potencial para lise e dissolução de biofilme da cepa Pseudomonas aeruginosa PA14, conhecida como agente causador de infecções hospitalares em pacientes imunodeprimidos. Em suma, os dados reunidos nesta dissertação caracterizam a diversidade presente no viroma da compostagem e contribuem para o entendimento dos perfis taxonômico, funcional e ecológico do processo. / The study of the viral diversity in environmental samples has become increasingly important due to key-roles that are performed by these organisms in our ecosystems. Recent publications provide evidence that viruses of bacteria (bacteriophages) may be key-players in biogeochemical cycles of large ecosystems, as oceans and forests. Besides, they may also be determinant in the genes flux among populations and in the plasticity of the communities face to environmental stresses. In this work, we propose the investigation and characterization of the viral diversity in composting samples through non-culturable and culturable-dependent approaches. In the first approach, we sampled a composting unit from the Sao Paulo Zoo Park in different time points and proceeded with metagenomic sequencing. The dataset generated was extensively mined to provide results of diversity and abundance of viral taxa through the composting process. Additionally, we proceeded with the assembly and retrieval of candidate sequences to partial or/and complete viral genomes. The two computational protocols were automatized as pipelines and can be applied to any metagenomic dataset of illumina reads. The second approach refers to the isolation and characterization of new Pseudomonas phages obtained from composting samples. Three new phages were identified and their genomes were sequenced. A detailed characterization of these genomes revealed high degree of novelty, insights about evolution of tailed-phages and the presence of tRNA genes, which may be related to a mechanism to bypass host translational bias. The experimental characterization of the new phages demonstrated great potential to lyse bacterial cells and to degrade Pseudomonas aeruginosa PA14 biofilms. In short, the data presented in this dissertation shed light to the composting virome diversity, as well as to the functional and ecological profiles of viruses in the composting environment.

O estudo da diversidade viral em amostras ambientais tem se tornado cada vez mais importante devido a funções-chave desempenhadas por esses organismos. Estudos recentes têm fornecido evidências de que vírus de bactérias (bacteriófagos) podem ser os principais determinantes em ciclos biogeoquímicos de grandes ecossistemas, além de atuarem no fluxo de genes entre comunidades ambientais e na plasticidade funcional das mesmas frente a estresses ambientais. Neste trabalho, propomos a investigação e caracterização da diversidade viral presente em amostras de compostagem através de abordagens não dependentes e dependentes de cultivo. Na primeira abordagem, coletamos amostras seriadas de uma unidade de compostagem do zoológico de São Paulo para realização de sequenciamento metagenômico. O conjunto de sequências gerado foi extensivamente minerado (data-mining) para a produção de resultados de diversidade e abundância de táxons virais ao longo do processo de compostagem. Adicionalmente, procedemos com a montagem e recuperação de sequências virais candidatas a genomas completos e/ou parciais de novos vírus ambientais. Os dois protocolos computacionais utilizados para a mineração de dados encontram-se definidos e automatizados, podendo ser aplicados em quaisquer conjuntos de dados de sequenciamento metagenômico ou metatranscritômico obtidos através da plataforma Illumina. A segunda abordagem correspondeu ao isolamento e caracterização de novos fagos de Pseudomonas obtidos de amostras de compostagem. Três novos fagos foram identificados e tiveram os seus genomas sequenciados. A caracterização genômica desses fagos revelou genomas com alto grau de novidade, insights sobre a evolução de Caudovirales e a presença de genes de tRNA, cuja função pode estar relacionada com um mecanismo dos fagos para contornar o viés traducional apresentado pela bactéria hospedeira. A caracterização experimental dos novos fagos isolados demonstrou grande potencial para lise e dissolução de biofilme da cepa Pseudomonas aeruginosa PA14, conhecida como agente causador de infecções hospitalares em pacientes imunodeprimidos. Em suma, os dados reunidos nesta dissertação caracterizam a diversidade presente no viroma da compostagem e contribuem para o entendimento dos perfis taxonômico, funcional e ecológico do processo. / The study of the viral diversity in environmental samples has become increasingly important due to key-roles that are performed by these organisms in our ecosystems. Recent publications provide evidence that viruses of bacteria (bacteriophages) may be key-players in biogeochemical cycles of large ecosystems, as oceans and forests. Besides, they may also be determinant in the genes flux among populations and in the plasticity of the communities face to environmental stresses. In this work, we propose the investigation and characterization of the viral diversity in composting samples through non-culturable and culturable-dependent approaches. In the first approach, we sampled a composting unit from the Sao Paulo Zoo Park in different time points and proceeded with metagenomic sequencing. The dataset generated was extensively mined to provide results of diversity and abundance of viral taxa through the composting process. Additionally, we proceeded with the assembly and retrieval of candidate sequences to partial or/and complete viral genomes. The two computational protocols were automatized as pipelines and can be applied to any metagenomic dataset of illumina reads. The second approach refers to the isolation and characterization of new Pseudomonas phages obtained from composting samples. Three new phages were identified and their genomes were sequenced. A detailed characterization of these genomes revealed high degree of novelty, insights about evolution of tailed-phages and the presence of tRNA genes, which may be related to a mechanism to bypass host translational bias. The experimental characterization of the new phages demonstrated great potential to lyse bacterial cells and to degrade Pseudomonas aeruginosa PA14 biofilms. In short, the data presented in this dissertation shed light to the composting virome diversity, as well as to the functional and ecological profiles of viruses in the composting environment.

This thesis focuses on conflict resolution and collective decision-making in co-navigating pigeons, Columba livia. These birds have a remarkable homing ability and frequently fly in flocks. Group navigation demands that group members reach consensus on which path to follow, but the mechanisms by which they do so remain largely unexplored. Pigeons are particularly suitable for studying these mechanisms, due to their sociality and the fact that their possession of information can easily be altered and quantified. I present the results of a series of experiments that manipulated the experience of homing pigeons in various ways so as to observe the effect of information they had previously gathered on their group behaviour. Key findings were: Previous navigational experience contributes to the establishment of leader-follower relationships. The larger the difference in experience between two co-navigating pigeons, the higher the likelihood the more experienced bird will emerge as leader. Shared homing experience through repeated joint flights can allow two pigeons to develop into a “behavioural unit”. They form spatial sub-groups when flying with less familiar birds, and perform a similar transition between compromise- and leadership-dominated flights as single birds, although they are more likely to accept compromise routes. Such previous association histories between birds can thus affect collective decision-making in larger flocks. There is a trade-off between the amount of spatial information handled and the efficiency with which such information can be applied during homing. Leading/following behaviour is influenced by the recency of the route memories. Leadership hierarchies in pigeon flocks appear resistant to changes in the navigational knowledge of a subset of their members, at least when these changes are relatively small in magnitude. The stability of the hierarchical structure might be beneficial during decision-making. Mathematical modelling suggests that underlying hierarchical social structures can increase navigational accuracy. Hierarchically organised groups with the smallest number of strong connections achieve highest accuracy. Group leader-follower dynamics resemble the underlying social structure.

Hematopoietic stem cells (HSCs) transplantations are widely used for the treatment of hematological and non-hematological disorders in clinic. Successful transplantation requires sufficient number and efficient homing of HSCs. Many studies have focused on developing an effective strategy to expand functional HSC population. Some regulatory molecules have been recently shown great promise for controlling the amplification of HSCs. In these dissertation studies, I first aim to identify gene(s) and their allelic variants contributing to strain-specific difference in HSC numbers between C57BL/6 (B6, low) and DBA/2 (D2, high) mice by using a classic forward genetic approach. Firstly, 3 quantitative trait loci (QTL) on chromosome (Chr) 3,5 and 18 were mapped by linkage analyses and confirmed in congenic mice. Secondly, Chr.3 QTL affected several HSC number-related biological processes. The D2 allele increased cycling and self-renewal whereas it decreased apoptotic rates of HSCs. Both actions conspired to increase HSC population size. Lastly, a small number of differentially-expressed genes was identified in Chr.3 congenic HSCs by a microarray-based candidate gene method, and the differential expression of one candidate, latexin, was found to relate to HSC number variations. Our studies report the strong evidence for the potential functions of latexin in HSC number regulation, and they are important for understanding molecular mechanisms of stem cell regulation and developing effective stem cell expansion strategies for clinical applications. In the second part of my studies, I studied homing and engraftment capabilities of HSCs. By using functional assays for progenitor and stem cells, I first reported the absolute homing efficiencies of murine young or old donor cells into young or old recipient mice. The results indicated that homing of primitive hematopoietic cells was not efficient and significantly decreased by aging of donors and recipients. The proliferation and differentiation states of HSCs were also impaired by homing itself, as well as by donors' and recipients age. Moreover, the hematopoietic reconstitution dynamics following transplantation were also affected by aging. Together, these findings will provide useful information for clinical applications especially when older individuals increasing serve as stem cell donors for elderly patients.

Acute myocardial infarction (AMI) triggers the mobilization of stem/progenitor cells from bone marrow (BMSPCs) into peripheral blood (PB). The underlying mechanisms orchestrating this mobilization and subsequent homing of BMSPCs to the myocardium are poorly understood. While the role of traditional chemokines in the mobilization and homing of hematopoietic stem cell (HSCs) to BM niches is undisputed, their role in directing BMSPCs to the highly proteolytic environment of the ischemic myocardium is debatable and other redundant mechanism may exist. Based on our observation that bioactive lipids, such as sphingosine-1 phosphate (S1P) and ceramide-1 phosphate (C1P), play an important role in regulating trafficking of HSCs; we explored if they also direct trafficking of BMSPCs in the setting of myocardial ischemia. While BMSPCs expressed S1P receptors regardless of the source, the expression of S1P receptor 1 (S1PR1) and receptor 3 (S1PR3), which are responsible for migration and chemotaxis, was elevated in BMSPCs in naïve BM cells and was reduced following mobilization. This expression correlated to differential response of BMSPCs to S1P in chemotaxis assays. By employing flow cytometry analyses, we observed an increase in circulating PB CD34+, CD133+ and CXCR4+ lineage negative (Lin-)/CD45- cells that are enriched in non-HSCs (P < 0.05 vs. controls). This corroborated our mass spectrometry studies showing a temporal increase in S1P and C1P plasma levels. At the same time, plasma obtained in the early phases following AMI strongly chemoattracted human BM-derived CD34+/Lin- and CXCR4+/Lin- cells in Transwell chemotaxis assays in an S1P dependent fashion. We examined other mechanisms that may contribute to the homing of BMSPCs to the infarcted myocardium due to the reduction of S1PRs upon mobilization. We observed that hypoxia induced higher expression of cathelicidins in cardiac tissues. Indeed, PB cells isolated from patients with AMI migrated more efficiently to low, yet physiological, gradient of SDF-1 in Transwell migration assays compared to SDF-1 alone. Together, these observations suggest that while elevated S1P plasma levels early in the course of AMI may trigger mobilization of non-HSCs into PB, cathelicidins appear to play an important role in their homing to ischemic and damaged myocardium.

This thesis investigates how collective decisions in bird flocks arise from simple rules, the factors that give some birds more influence over a flock's direction, and how travelling as a flock affects spatial learning. I used GPS loggers to track pigeons homing alone and in flocks, and applied mathematical modelling to explore the mechanisms underlying group decisions. Across several experiments, the key results were as follows: Flying home with a more experienced individual not only gave a pigeon an immediate advantage in terms of taking a more direct route, but the followers also learned homing routes just as accurately as pigeons flying alone. This shows that using social cues did not interfere with learning about the landscape during a series of paired flights. Pigeons that were faster during solo homing flights also tended to fly at the front of flocks, where they had more influence over the direction taken. Analysis of momentary interactions during paired flights and simulations of pair trajectories support the conclusion that speed increases the likelihood of leading. A pigeon's solo homing efficiency before flock flights did not correlate with leadership in flocks of ten, but leaders did have more efficient solo tracks when tested after a series of flock flights. A possible explanation is that leaders attended more to the landscape and therefore learned faster. Flocks took straighter routes than pigeons flying alone, as would be expected if they effectively pooled information. In addition, pigeons responded more strongly to the direction of several neighbours, during flock flights, than to a single neighbour during paired flights. This behaviour makes sense adaptively because social information will be more reliable when following several conspecifics compared to one. Through a combination of high-resolution tracking and mathematical modelling, this thesis sheds light on the mechanisms of flocking and its navigational consequences.

Researches on Hematopoietic Stem Cell (HSC) have been expanding that leads to an increase in our understanding of HSC normal behaviors and abnormal alterations. One of the most important issues in the research on HSCs is to understand the mechanism of the homing process of these cells to settle in their niche in the bone marrow and establish the production of various blood cell types after bone marrow transplantation. The cells first must come in contact with the endothelial cells. This contact is known as adhesion and occurs through a multi-step paradigm ending with transmigration to the bone marrow niche. The initial step of the homing, tethering and rolling of HSC, is mediated by P- and E-Selectins present on endothelial cell surface through their interactions with the ligands expressed on the surface of HSC. Thus, understanding the adhesion process and its contribution for efficient HSCs homing will have great impact on HSC therapy. The selectin – ligands interaction has been intensively studied using in vivo and in vitro approaches. However, the molecular mechanism involved by HSCs at single molecule level is poorly understood. Here in this study, a novel experimental method to unravel the molecular mechanisms of the Selectin-ligands interactions in vitro at the single molecule level is developed by combining microfluidics, epi-fluorescence microscopy and live cells. In this work, the new single-molecule imaging technique enabled us to directly visualize the nanoscale spatiotemporal dynamics of the membrane protein-ligand interactions under conditions of shear stress acting on the cells at the molecular level in real time. Using this method, we revealed that selectin ligands on membrane-tethers and slings show unique spatiotemporal dynamics that is distinct from those on the cell body. We demonstrated that the membrane tethers are formed from single microvilli on the cells, which provides a mechanism to spatially localize selectin ligands, PSGL-1 and CD44 on the tethers and slings. We also demonstrated that the selectin ligands show fast diffusional motion along the tethers and slings compared with that on the cell body due to the detachment of cell membranes from actin cytoskeleton during the formation of the tethers. Our results suggest that the spatial confinement of the selectin ligands together with the fast scanning of a large area by the selectin ligands increase the efficiency of selectin-ligands interaction during the rolling, resulting in slow and stable rolling of the cell on selectin. Our findings contribute significantly to molecular level understanding of the initial step of HSCs. This single-molecule imaging technique that we developed in this study will find wide applications in the molecular-level studies on cell-cell interactions including cancer cell metastasis.