Epigenetics is the study of heritable biological variation not related to changes in DNA sequence. Epigenetic processes are responsible for establishing and maintaining transcriptional programs that define cell identity. Defects to epigenetic processes have been linked to a host of disorders, including mental retardation, aging, cancer and neurodegenerative diseases. The ability to control and engineer epigenetic systems would be valuable both for the basic study of these critical cellular processes as well as for synthetic biology. Indeed, while synthetic biology has made progress using bottom-up approaches to engineer transcriptional and signaling circuitry, epigenetic systems have remained largely underutilized. The predictive engineering of epigenetic systems could enable new functions to be implemented in synthetic organisms, including programmed phenotypic diversity, memory, reversibility, inheritance, and hysteresis. This thesis broadly focuses on the development of foundational tools and intellectual frameworks for applying synthetic biology to epigenetic regulation in the model eukaryote, Saccharomyces cerevisiae. Epigenetic regulation is mediated by diverse molecular mechanisms: e.g. self-sustaining feedback loops, protein structural templating, modifications to chromatin, and RNA silencing. Here we develop synthetic tools and circuits for controlling epigenetic states through (1) modifications to chromatin and (2) self-templating protein conformations. On the former, the synthetic tools we develop make it possible to study and direct how chromatin regulators operate to produce distinct gene expression programs. On the latter, we focus our studies on yeast prions, which are self-templating protein conformations that act as elements of inheritance, developing synthetic tools for detecting and controlling prion states in yeast cells. This thesis explores the application of synthetic biology to these epigenetic systems through four aims: Aim 1. Development of inducible expression systems for precise temporal expression of epigenetic regulators Aim 2. Construction of a library of chromatin regulators to study and program chromatin-based epigenetic regulation. Aim 3. Development of a genetic tool for quantifying protein aggregation and prion states in high-throughput Aim 4. Dynamics and control of prion switching Our tools and studies enable a deeper functional understanding of epigenetic regulation in cells, and the repurposing of these systems for synthetic biology toward addressing industrial and medical applications. / 2019-10-08T00:00:00Z

Desde o início do século XXI, vivenciamos uma explosão na produção de informações de diversos tipos, tais como fotos, áudios, vídeos, entre outros. Dentre essas informações, existem aquelas em que a informação pode ser dividida em partes menores, mas que devem ser relacionadas seguindo uma ordem total. Um exemplo deste tipo de informação é um arquivo de vídeo que foi dividido em dez segmentos identificados com números de 1 a 10. Para reproduzir o vídeo original a partir dos segmentos é necessário que seus identificadores estejam ordenados. A estrutura denominada tabela de hash distribuída (DHT) tem sido amplamente utilizada para armazenar, atualizar e recuperar esse tipo de informação de forma eficiente em diversos cenários, como monitoramento de sensores e vídeo sob demanda. Entretanto, a DHT apresenta problemas de escalabilidade quando um membro da estrutura não consegue atender as requisições recebidas, trazendo como consequência a inacessibilidade da informação. Este trabalho apresenta uma arquitetura em camadas denominada MATe, que trata o problema da escalabilidade em dois níveis: estendendo a DHT com a introdução de agentes baseados na utilidade e organizando a quantidade de requisições solicitadas. A primeira camada trata a escalabilidade ao permitir a criação de novos agentes com o objetivo de distribuir as requisições evitando que um deles tenha a escalabilidade comprometida. A segunda camada é composta por grupos de dispositivos organizados de tal forma que somente alguns deles serão escolhidos para fazer requisições. A arquitetura foi implementada para dois cenários onde os problemas de escalabilidade acontecem: (i) monitoramento de sensores; e (ii) vídeo sob demanda. Para ambos cenários, os resultados experimentais mostraram que MATe melhora a escalabilidade quando comparada com as implementações originais da DHT. / Since the beginning of the 21st century, we have experienced an explosive growth in the generation of information, such as photos, audios, videos, among others. Within this information, there are some in which the information can be divided and related following a total order. For example, a video file can be divided into ten segments identified with numbers from 1 to 10. To play the original video from these segments, their identifiers must be fully ordered. A structure called Distributed Hash Table (DHT) has been widely used to efficiently store, update, and retrieve this kind of information in several application domains, such as video on demand and sensor monitoring. However, DHT encounters scalability issues when one of its members fails to answer the requests, resulting in information loss. This work presents MATe, a layered architecture that addresses the problem of scalability on two levels: extending the DHT with the introduction of utility-based agents and organizing the volume of requests. The first layer manages the scalability by allowing the creation of new agents to distribute the requests when one of them has compromised its scalability. The second layer is composed of groups of devices, organized in such a way that only a few of them will be chosen to perform requests. The architecture was implemented in two application scenarios where scalability problems arise: (i) sensor monitoring; and (ii) video on demand. For both scenarios, the experimental results show that MATe improves scalability when compared to original DHT implementations.

Embryonic haematopoiesis is a complex process under intensive research. Murine definitive Haematopoietic Stem Cells (HSCs) originates from the Aorta-Gonad-Mesonephros (AGM) region of E10.5 embryo. It is thought that definitive HSCs arise from endothelial lining of dorsal aorta. However, detail of HSC specification in the developing embryo remains elusive. One way to deciphering events occurred during HSC specification is to derive cell lines from the developing HSC niche. Previous work by Oostendorp et al. showed the AGM and fetal liver derived lines could maintain HSCs in vitro (Oostendorp, Harvey et al. 2002). In this study, I established a more robust immortalization system using normal SV40 large T antigen delivered via Neon™ electroporation system. The new immortalization system achieved direct immortalization without going through crisis. And it is compatible with small number of primary cells dissected from different haematopoietic niches. With my new system, multiple cell lines from different haematopoietic sites at different developmental points are derived. Moreover, some of these lines demonstrated ability to mature precursors from E9.5 embryo (pro-HSCs) to definitive HSC without help of growth factors. This result is better compared to OP9 stromal lines. Such data proved usefulness of using stromal cell lines to study haematopoietic specification.

O crescente volume de informações disponíveis na rede mundial de computadores, gera a necessidade do uso de ferramentas que sejam capazes de localizá-las e ordenálas, de forma cada vez mais precisa e que demandem cada vez menos recursos computacionais. Esta necessidade tem motivado pesquisadores a estudar e desenvolver modelos e técnicas que atendam esta demanda. Estudos recentes têm sinalizado que utilizar vários ordenamentos (rankings) previamente montados possibilita o retorno e ordenação de objetos de qualquer natureza com mais eficiência, principalmente pelo fato de haver uma redução no custo da busca pela informação. Este processo, conhecido como fusão de rankings, permite que se obtenha um ordenamento com base na opinião de diversos juízes (critérios), o que possibilita considerar um grande número de fontes, tanto geradas automaticamente como por especialistas. Entretanto os modelos propostos até então tem apresentado várias limitações na sua aplicação: desde a quantidade de rankings envolvidos até, principalmente, a utilização de rankings parciais. A proposta desta dissertação é apresentar um modelo de fusão de rankings que busca estabelecer um consenso entre as opiniões (rankings) dos diferentes juízes envolvidos, considerando distintos graus de relevância ou importância entre eles. A base desta proposta está na Análise de Preferência, um conjunto de técnicas que permite o tratamento da multidimensionalidade dos dados envolvidos. Ao ser testado em uma aplicação real, o modelo mostrou conseguir suprir algumas limitações apresentadas em outras abordagens, bem como apresentou resultados similares aos das aplicações originais. Esta pesquisa, ainda contribui, com a especificação de um sistema Web baseado em tecnologias open source, o qual permite que qualquer pessoa possa realizar a fusão de rankings. / The growing volume of available information on the web creates the need to use tools that are capable of retrieve and ordering this information, ever more precise and using less computer resources. This need has motivated researchers to study and develop models and techniques that solve this problem. Recent studies have indicated that use multiple rankings previously mounted makes possible the return and sorting of the objects of any kind with more efficiency, mainly because there is a reduction in the cost of searching for information. This process, called ranking fusion, provide a ranking based on the opinion of several judges (criteria), considering a large number of sources, both generated automatically and also by specialists. However the proposed models have shown severe limitations in its application: from the amount involved rankings to the use of partial rankings. The proposal of this dissertation is to show a model of ranking fusion that seeks to establish a consensus between the judgement (rankings) of the various judges involved, considering different degrees of relevance or importance among them. The baseline of this proposal is the Preference Analysis, a set of techniques that allows the treatment of multidimensional data handling. During tests in a real application, the model supplied some limitations presented by other approaches, and presented results similar to the original applications. Additionally, this research contributes with the specification of a web system based on open-sources technologies, enabling the realization of fusion rankings by anyone.

O presente trabalho teve por objetivo compreender sistemas de produção agrícola e agropecuária a campo pela avaliação da diversidade, funcionalidade e dinâmica espaço-temporal de espécies, assim como pelo padrão de variação da qualidade do solo e dos fatores que a determinam. Para isso, foi avaliado na “Agropecuária Guajuvira” localizada no município de São Miguel das Missões – RS quatro sistemas conservacionistas de produção. Foram eles: 1- sistema agrícola tradicional, representando a sucessão soja/trigo e soja/aveia preta amplamente praticada na região; 2- Sistema agrícola irrigado, semelhante ao anterior, mas, com recente inserção de milho no verão; 3- Sistema integrado de produção agropecuária 1, representando a sucessão soja/pastejo de azevém e 4- Sistema integrado de produção agropecuária 2, representando um sistema misto por apresentar alterações na composição de espécies no inverno pela sucessão soja/aveia preta pastejada, soja/aveia preta não pastejada e soja/nabo forrageiro/trigo O estoque de carbono (EC), estabilidade de agregados do solo (EAS) e índice de manejo de carbono (IMC) foram escolhidos como indicadores da qualidade sistêmica do solo. Seus padrões de variação foram compreendidos pela integração de atributos químicos, físicos e biológicos do solo, assim como por variáveis de paisagem inerentes às unidades amostrais. Os fatores que caracterizaram os sistemas de produção e o uso da análise de caminhos permitiram um maior entendimento de sistemas complexos de produção agrícola e agropecuária a campo. / The aim of this research was to understand agricultural and integrated crop-livestock production systems by the species diversity, functionality and spatial-temporal dynamics evaluation, as well as of the variation pattern of soil quality and factors that determine then. For this, four no-tillage production systems were evaluated on the "Agropecuária Guajuvira" located in São Miguel das Missões county in southern Brazil. The production systems were: 1- Traditional agricultural system, representing the succession soybean/wheat and soybean/black oat widely practiced in the region; 2- Irrigated agricultural system, similar to the previous one, but, with recent insertion of corn in the summer; 3 - Integrated crop-livestock system 1, representing the succession of soybean /grazed ryegrass and 4 - Integrated crop-livestock system 2, representing a mixed system due changes in species composition during winter by succession of soybean/grazed black oat, soybean/no-grazed black oat and soybean/forage radish/wheat The carbon stock (CS), soil aggregate stability (SAS) and carbon management index (CMI) were chosen as systemic soil quality indicators. Their variation patterns were understood by the integration of chemical, physical and biological soil attributes, as well as by landscape variables inherent to the sampling units. The factors that characterized the production systems and the path analysis utilization allowed a greater understanding of complex agricultural and integrated crop-livestock production systems in the field.

In this thesis, we investigate the mechanisms driving the self-assembly of peptides and proteins using computational and theoretical tools, always validating our results with experimental measures when possible. In the first part, Chapters 2-5, we focus on the Aβ system, a peptide whose aggregation is intimately linked with the development of Alzheimer's Disease. We begin by simulating the major alloforms of the peptide, Aβ_40 and Aβ_42, demonstrating that the two populate similar disordered ensembles and matching experimental data. Next we investigate how disordered Aβ_42 monomers interact with each other, finding that oligomerisation into amorphous aggregates is driven largely by hydrophobic, non-specific forces. We then move on to probing the aggregation of Aβ_42 into amyloid structures using a native-centric coarse-grained model, and explain the results with a novel Markov state analysis from which we are able to extract structural, kinetic and thermodynamic information on elongation reactions. Finally, we probe the interactions of Aβ_42 monomers with Aβ_42 fibrillar surfaces using a specially designed enhanced sampling scheme, which allows us to obtain enthalpy-driven binding thermodynamics consistent with experiments and to propose major polar binding modes. In the second part of the thesis, Chapters 6 and 7, we model the aggregation of two other self-assembling systems, viruses and a truncated form of the molecular chaperone Hsp70. We first develop a data analysis platform to extract information on the microscopic mechanisms of viral capsid self-assembly from experimental data, synthesising the results from several different systems to draw general evolutionary conclusions about the assembly mechanism. Finally, we model the oligomerisation of Hsp70 thermodynamically and kinetically, showing that its self-assembly is a highly cooperative reaction that is under strong structural constraints.

Made available in DSpace on 2014-06-11T19:35:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-12-17Bitstream added on 2014-06-13T20:25:43Z : No. of bitstreams: 1 souza_se_dr_botfca.pdf: 819772 bytes, checksum: f7cd95ae9d4f5c4dc21861b150059d3e (MD5) / Em três povoamentos de Eucalyptus grandis na região norte do Estado de São Paulo, foi realizado um estudo com objetivo de conhecer a dinâmica espaço-temporal do cancro basal, quantificar os danos no volume e efeito na qualidade da madeira. Foram instaladas nove parcelas de 7500 m², com 1000 árvores de E. grandis, procedência Santa Rita do P. Quatro-SP, origem Coffs Harbour, dos plantios seminais em solos de areia franca AQ1, AQ3 (10 a 15% de argila) e textura franco-argila-arenosa LEm2 (26 a 35% de argila). As avaliações foram realizadas em nove mil árvores com idade de dois anos. A incidência foi monitorada individualmente de acordo com a presença ou ausência da doença e a severidade, mediante o uso de uma escala diagramática de notas. Foram feitas avaliações nos anos 2000, 2001 e 2002, o que gerou 26 mapas de evolução da doença. Os resultados permitiram concluir que: (1) A incidência média do cancro basal foi influenciado pelo tipo de solo AQ3 (0,25%), AQ1 (0,21%) e LEm2 (0,09%); (2) Os valores do índice de dispersão (ID) foram > 1, para os tamanhos de quadrat 2x4, 5x2, 5x4 e 5x10; os valores de log (A) = log (Vobs) e log de (b) = log (Vbin) foram maiores que 1 e diferentes de zero, sugerindo uma tendência à agregação independente do tipo de solo; (3) 54,6% dos focos foram unitários, indicando que o cancro basal inicia-se com focos de apenas uma árvore; 80,8% dos focos apresentam maior comprimento na direção entre as linhas de plantio do que na direção da linha; focos maiores ocorreram no solo AQ3, que apresentaram-se menos compactos; a disseminação do cancro nesses povoamentos parece ter sido via sementes através das mudas; 89,1% dos mapas apresentaram focos com tamanho médio inferior a 10 árvores, no solo AQ3 em 10,9% dos focos o tamanho variou entre 11 a 30 árvores e nenhum foco foi formado acima de 30 árvores;... / A study was carried out in three Eucalyptus grandis stands in the northern region of the State of São Paulo, in order to provide information on the spatio-temporal dynamics of eucalyptus canker, evaluate volume losses, and determine the effect of the disease on wood quality. Nine 7500 m² plots (10×100) containing 1000 E. grandis trees from Santa Rita do P. Quatro-SP (origin: Coffs Harbour), in seed plantations on soils classified as loamy sands, AQ1 and AQ3 (10 to 15% clay), and on a silty-clay-loam-textured soil, LEm2 (26 – 35% clay). Evaluations were made in nine thousand two-year-old trees. Incidence was monitored individually according to the presence or absence of the disease. Severity was evaluated through the use of a diagrammatic rating scale. Evaluations were made in 2000, 2001, and 2002, and 26 disease progress maps were generated. The results allowed us to conclude that: (1) The mean incidence of eucalyptus canker followed a trend that matched the soil types:AQ3 (0.25%), AQ1(0.21.%), and LEm2 (0.09%); (2) The ID values were > 1 for quadrat sizes equal to 2×4, 5×2, 5×4, and 5×10; The log (A) = log (Vobs) and log (b) = log (Vbin) values were higher than 1 and different from zero, suggesting a tendency for aggregation, regardless of soil type; (3) 54.6% of the foci were single-unit, indicating that eucalyptus canker starts with single-tree foci; 80.8% of the foci had greater lengths between planting rows than along the rows; larger foci occurred in the AQ3 soil, which were less compact; canker dissemination in those stands seemed to have occurred via seeds through the seedlings; 89.1% of the maps had foci with mean sizes smaller than 10 trees in the AQ3 soil; in 10.9% of the foci the size ranged between 11 and 30 trees, and no focus formed at sizes above 30 trees; the ICF and ICFNU values were not close nor very far from ...(Complete abstract click electronic access below)

O crescente volume de informações disponíveis na rede mundial de computadores, gera a necessidade do uso de ferramentas que sejam capazes de localizá-las e ordenálas, de forma cada vez mais precisa e que demandem cada vez menos recursos computacionais. Esta necessidade tem motivado pesquisadores a estudar e desenvolver modelos e técnicas que atendam esta demanda. Estudos recentes têm sinalizado que utilizar vários ordenamentos (rankings) previamente montados possibilita o retorno e ordenação de objetos de qualquer natureza com mais eficiência, principalmente pelo fato de haver uma redução no custo da busca pela informação. Este processo, conhecido como fusão de rankings, permite que se obtenha um ordenamento com base na opinião de diversos juízes (critérios), o que possibilita considerar um grande número de fontes, tanto geradas automaticamente como por especialistas. Entretanto os modelos propostos até então tem apresentado várias limitações na sua aplicação: desde a quantidade de rankings envolvidos até, principalmente, a utilização de rankings parciais. A proposta desta dissertação é apresentar um modelo de fusão de rankings que busca estabelecer um consenso entre as opiniões (rankings) dos diferentes juízes envolvidos, considerando distintos graus de relevância ou importância entre eles. A base desta proposta está na Análise de Preferência, um conjunto de técnicas que permite o tratamento da multidimensionalidade dos dados envolvidos. Ao ser testado em uma aplicação real, o modelo mostrou conseguir suprir algumas limitações apresentadas em outras abordagens, bem como apresentou resultados similares aos das aplicações originais. Esta pesquisa, ainda contribui, com a especificação de um sistema Web baseado em tecnologias open source, o qual permite que qualquer pessoa possa realizar a fusão de rankings. / The growing volume of available information on the web creates the need to use tools that are capable of retrieve and ordering this information, ever more precise and using less computer resources. This need has motivated researchers to study and develop models and techniques that solve this problem. Recent studies have indicated that use multiple rankings previously mounted makes possible the return and sorting of the objects of any kind with more efficiency, mainly because there is a reduction in the cost of searching for information. This process, called ranking fusion, provide a ranking based on the opinion of several judges (criteria), considering a large number of sources, both generated automatically and also by specialists. However the proposed models have shown severe limitations in its application: from the amount involved rankings to the use of partial rankings. The proposal of this dissertation is to show a model of ranking fusion that seeks to establish a consensus between the judgement (rankings) of the various judges involved, considering different degrees of relevance or importance among them. The baseline of this proposal is the Preference Analysis, a set of techniques that allows the treatment of multidimensional data handling. During tests in a real application, the model supplied some limitations presented by other approaches, and presented results similar to the original applications. Additionally, this research contributes with the specification of a web system based on open-sources technologies, enabling the realization of fusion rankings by anyone.

Due to the high availability of cheap hardware, wireless multi-hop networks and in particular Wireless Mesh Networks (WMNs) are becoming popular in more and more contexts. For instance, IEEE 802.11 based WMNs have already started to be deployed as means to provide Internet access to rural areas in the developing world. To lower the cost and increase the coverage in such deployments, the wired network is extended with a wireless backbone of fixed mesh routers. With advances in technology and reduction in price comes also the possibility for more powerful wireless nodes, having multiple radios that allow transmitting on different channels in parallel. To be a successful platform for providing general Internet access, wireless multi-hop networks must provide support for common Internet applications. As most of the applications in the Internet today use the Transmission Control Protocol (TCP), TCP performance is crucial. Unfortunately, the design of TCP’s congestion control that made it successful in today’s Internet makes it perform less than optimal in wireless multi-hop networks. This is due to, among others, TCP’s inability to distinguish wireless losses from congestion losses. The current trend for operating system designers is also to focus TCP development on high-speed fixed networks, rather than on wireless multi-hop networks. To enable wireless multi hop networks as a successful platform there is therefore a need to provide good performance using TCP variants commonly deployed in the Internet. In this thesis, we develop novel proposals for the network layer in wireless multi-hop networks to support TCP traffic more efficiently. As an initial study, we experimentally evaluate different TCP variants, with and without mobile nodes, in a MANET context. Our results show that TCP Vegas, which does not provoke packet loss to determine available bandwidth, reduces the stress on the network while still providing the same or slightly increased performance, compared to TCP Newreno. We further propose and evaluate packet aggregation combined with aggregation aware multi-path forwarding to better utilize the available bandwidth. IP layer packet aggregation, where small packets are combined to larger ones before sent to the link layer, has been shown to improve the performance in wireless multi-hop networks for UDP and small packet transfers. Only few studies have been made on the impact of packet aggregation on TCP traffic, despite the fact that TCP traffic constitutes the majority of the Internet traffic. We propose a novel aggregation algorithm that is specifically addressing TCP relevant issues like packet reordering, fairness and TCP timeouts. In a typical WMN scenario, the aggregation algorithm increases TCP performance by up to 70 % and decreases round trip time (RTT) by up to 40 %. A detailed evaluation of packet aggregation in a multi radio setting has shown that a naive combination of multi path routing and packet aggregation can cause valuable aggregation opportunities to be lost. Therefore, we propose a novel combined packet aggregation and aggregation aware forwarding strategy that can reduce delay, packet loss and increase TCP performance by around 30 %.

The aim of this work is to prepare heterogeneous nanoparticles which means nanoparticles composed of more than one material. Our approach lies in in-flight modification of primary nanoparticles in the tubular sputtering system. Our tubular system contains copper target and we deposit copper onto the flying primary nanoparticles by magnetron sputtering. The main advantage of this approach is independence of fabrication of primary nanoparticles and their subsequent modification. At first we optimized fabrication of nanoparticles by the gas aggregation source on behalf of the next modification. We also characterized conditions in the tubular sputtering system. We found process in the tubular system to be very complex and sensitive to the changes of the operational parameters. There is a strong interaction between flying nanoparticles and the discharge in the tubular system. Due to this interaction the nanoparticles are trapped in the plasma and the deposition rate is pulsing. The result of this work is modification of nickel and silver nanoparticles, preparation of heterogeneous nanoparticles Ni/Cu and Ag/Cu. These heterogeneous nanoparticles vary in composition, shape and size according to the conditions in the tubular system. We also successfully prepared Janus nanoparticles which are interesting for their...