Thesis (MScAgric)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Plant carotenoid cleavage dioxygenases (CCD) are a family of enzymes that catalyse the oxidative cleavage of carotenoids and/or apocarotenoids. Carotenoids are synthesised in plastids (primarily chloroplasts and chromoplasts), where they are involved in light-harvesting and protecting the photosynthetic apparatus from photo-oxidation. The carotenoid-derived apocarotenoids fulfil a number of roles in plants such as phytohormones, pollinator attractants and flavour and aroma compounds. Due to the floral and fruity characteristics that apocarotenoids contribute to wine, these C13 compounds have received interest in grapevine (Vitis vinifera L.). The CCD gene family in Arabidopsis consists of nine members, all encoding for enzymes that catalyse the cleavage of carotenoids. The enzymes in this family include 9-cis-epoxydioxygenases (NCEDs) and four classes of CCD. NCEDs and CCD7 and CCD8 are involved with plant hormone synthesis, e.g. abscisic acid (ABA) through cleavage by NCED and strigolactone (SL) through the sequential cleavage of carotenoids by CCD7 and CCD8, respectively. SLs are a fairly new class of plant hormone which are involved in several aspects of plant growth and development. The most extensively characterised role of SLs is their involvement in the inhibition of shoot-branching. CCD1 and CCD4 cleave a variety of carotenoids to form pigments and aroma compounds. For example, CCD1 forms β-ionone and β-damascenone, which are important varietal flavours of wine, and CCD4 is involved in synthesis of the pigment and aroma compounds of saffron and annatto. CCD1 enzymes symmetrically cleave the 9,10 (9’,10’) double bonds of multiple carotenoids to produce a C14 dialdehyde and two C13 products. Additional CCD1 cleavage activity at 5,6 (5’,6’) double bonds of lycopene has been reported. Previous studies have shown that CCD1 isolated from V. vinifera (VvCCD1) was able to cleave multiple carotenoid substrates in vitro, namely zeaxanthin, lutein and β-carotene at 9,10 (9’,10’) double bonds and both the 5,6 (5’,6’) and 9,10 (9’,10’) double bonds of lycopene. None of the other VvCCDs, except VvCCD4a have been isolated (but no functionality was illustrated) and characterised yet. CCD4 enzymes also cleave carotenoids at the 9,10 (9’,10’) double bond positions. The presence of plastid-target peptides implies that the CCD4 enzymes have continuous access to carotenoids. Therefore it is suggested that CCD4s are responsible for carotenoid maintenance, where CCD1s contribute towards volatile production. To test this hypothesis VvCCD1, VvCCD4a and VvCCD4b were isolated from V. vinifera (cv Pinotage) cDNA and cloned into a pTWIN1 protein expression vector. Substrate specificity of each VvCCD was tested by co-transforming a carotenoid accumulating E. coli strain with a CCD expression vector. Carotenoids synthesized by the bacteria were identified and quantified by UPLC-analysis, while the concentration of the apocarotenoids, were measured in the headspace of the bacterial cultures using HS-SPME-GC-MS. Several optimisations were done to minimize the natural degradation of the carotenoids; to ensure that the apocarotenoid formation is predominantly due to the enzymatic cleavage by the VvCCDs and not due to oxidation or other non-enzymatic degradation. The HS-SPME-GC-MS analysis indicated that all isoforms cleaved phytoene, lycopene and ε-carotene. Additionally VvCCD1 cleaved a carotenoid involved in photosynthesis, namely β-carotene, while VvCCD4a cleaves neurosporene and VvCCD4b cleaves neurosporene and ζ-carotene, carotenoids not involved in photosynthesis. This study has illustrated that VvCCD1 cleave carotenoids necessary for photosynthesis and VvCCD4s cleave carotenoids which were not present in berry tissue, suggesting their role in carotenoid maintenance. Therefore in planta substrates for CCD1 could possibly be C27 apocarotenoids generated from enzymatic cleavage through CCD4 (role in carotenoid maintenance), CCD7 and/or photo-oxidation, which are then transported from the plastid to the cytosol or possibly C40 carotenoids that are released during senescence or when the plastid membrane is damaged, thus releasing important aroma compounds. Thus the identification of the in vivo substrates has contributed to the understanding the in planta functions of these enzymes / AFRIKAANSE OPSOMMING: Die plant ensiemfamilie van karotenoïedsplitsingdioksigenases (CCDs) kataliseer die oksidatiewe splitsing van karotenoïede en/of apokarotenoïede. Karotenoïede word in plastiede (primêr chloroplaste en chromoplaste) sintetiseer en is betrokke by lig-absorpsie en die beskerming van die fotosintetiese apparaat teen foto-oksidasie. Die apokarotenïede afkomstig van karotenoïede dien onder meer as planthormone, geur- en aromakomponente en om bestuiwers aan te lok. Aangesien apokarotenoïede bydra tot die vrug- en blomgeure van wyn is die C13-verbindings binne wingerd (Vitis vinifera L.) van belang. Al nege lede van die CCD geenfamilie in Arabidopsis kodeer karotenoïedsplitsingsensieme. Die ensiemfamilie sluit 9-sis-epoksidioksigenases (NCEDs), en vier klasse CCD in. NCEDs en CCD7 en 8 is betrokke by die sintese van planthormone, naamlik absissiensuur (ABA) deur NCED en strigolaktone (SL) deur die opeenvolgende aksie van onderskeidelik CCD7 en CCD8. SLe is redelik onlangs as planthormone indentifiseer en is betrokke by ‘n verskeie aspekte van die groei en ontwikkeling van plante. Die rol van SL in inhibisie van vertakking is die beste gekarakteriseerde van hierdie aspekte. CCD1 en CCD4 splits ‘n verskeidenheid karotenoïede om pigmente en aromakomponente te vorm. CCD1 vorm byvoorbeeld β-jonoon en β-damasenoon, beide belangrike kultivar-spesifieke wyngeure. CCD4 vorm weer die pigment en aromakomponente van saffraan en annatto. Die CCD1 ensieme splits die 9,10 (9’,10’) dubbelbindingsetels van verskeie karotenoïede simmetries en vorm een C14-dialdehied en twee C13-produkte. Daar is voorheen melding gemaak van verdere splitsing deur CCD1 by die 5,6 (5’,6’) dubbelbindingsetels van likopeen. Vroeër is getoon dat die CCD1 isovorm wat uit V. vinifera geïsoleer is, naamlik VvCCD1, in vitro seaxantin, luteïen en β-karoteen by die 9,10 (9’,10’) dubbelbindingsetels kon splits, en likopeen by beide die 9,10 (9’,10’) en 5,6 (5’,6’) dubbelbindingsetels. Geen ander VvCCDs is al isoleer en funksioneel gekarakteriseer. VvCCD4a is isoleer, maar geen funksie is bepaal nie. CCD4 ensieme splits ook die 9,10 (9’,10’) dubbelbindingsetels van karotenoïede. Aangesien CCD4 ensieme ‘n plastied-bestemmingspeptied besit behoort dié ensieme konstant toegang tot karotenoïede te hê, wat dui op hul rol in die handhawing van die karotenoïedbalans, terwyl CCD1-ensieme bydra tot die sintese van vlugtige verbindings. Om hierdie hipotese te toets is VvCCD1, VvCCD4a en VvCCD4b uit V. vinifera (kv Pinotage) kDNS isoleer in binne ‘n pTWIN1 proteïenuitdrukkingsvektor kloneer. Die substraatspesifisiteit van elke VvCCD is getoets deur ‘n karotenoïedakkumulerende E. coil stam te transvormeer met ‘n CCD-uitdrukkingsvektor. UPLC-analise is gebruik om karotenoïede wat deur die bakterium sintetiseer is te kwantifiseer en identifiseer, terwyl die apokarotenoïedinhoud en -konsentrasie van die boruimte van die bakteriële kultuur met HS-SPME-GC-MS bepaal is. Verskeie aspekte van die proses is optimaliseer om natuurlike afbreking van karotenoïede te minimeer. Daardeur is verseker dat die apokarotenoïedvorming primêr vanweë die ensiematiese splitsing deur VvCCDs plaasvind en nie deur oksidasie of ander nie-ensiematiese afbreking. Die HS-SPME-GC-MS metings het aangedui dat al drie isovorme fitoëen, likopeen en ε-karoteen kan splits. VvCCD1 kan daarby β-karoteen splits, terwyl VvCCD4a neurosporeen, en VvCCD4b neurosporeen en ζ-karoteen kan splits, beide karotene wat nie betrokke is by fotosintese nie. Dié studie toon dat VvCCD1 die karotenoïede splits wat benodig word vir fotosintese, terwyl beide VvCCD4 isovorme karotenoïede splits wat nie in druiwekorrels gevind word nie. Dit dui op hulle rol in die handhawing van karotenoïedpoele. Die in planta substrate vir CCD1 mag dus die C27-apokarotenoïede wees wat deur CCD4 (as deel van karotenoïedhandhawing), CCD7 en/of foto-oksidasie gevorm word en na die sitosol vervoer word, of moontlik die C40-karotenoïede wat tydens veroudering óf wanner die plastiedmembraan beskadig is in die sitosol vrygestel word. Die identifisering van die in vivo substrate het dus bygedra to die begrip van die in planta funksies van die ensieme.

For milennia, agricultural genetics were common-pool, open access (or res nullius) resources, unencumbered by the assignment of property rights. Beginning in 1930, a series of legislative and judicial actions incrementally altered the legal definition of agricultural genetics and, ultimately, permitted the application of utility patents to a resource that was once free to all. In the factitious process of creating ownable property from that which was previously shared, the potential consequences of privatizing these res nullius resources were often unanticipated, underappreciated or entirely dismissed. Ramifications include not only the widely publicized concerns of environmental contretemps and the potentially insalubrious effects of consuming transgenic foods, but also more obscure implications, many of which are economically counterproductive and socially undesirable (e.g., promoting farmers onto a technology treadmill that requires them to implement successive iterations of evolving technologies or risk becoming noncompetitive; creating hostilities between technology adopters and non-adopting farmers which, in turn, complexifies social relationships and diminishes the quality of rural life; encouraging questionable corporate behaviors; promoting strategic hold-ups whereby broadly applied patents constrain the widespread use of licenses and consequently inhibit further evolution of technologies, and; creating patent thickets that produce bottlenecks, slow innovation, and increase transaction costs). Proponents of biotechnology seek to strengthen the inchoate property to the point of adoption by minimizing or negating controversial aspects while emphasizing potentially positive outcomes. Opposing interests attempt to exploit potentially negative implications or outcomes in an attempt to weaken the propertization to the point of abandonment. (According to Radin (2000b), the term, propertization, refers to the creation of property, often intangible, or at least less tangible than traditional chattel assets, through a socially sanctioned, uncertain and malleable process.) The success of any attempt to privatize agricultural genetics is not assured, and opportunistic stakeholders opposing or promoting the creation of the property will attempt to exploit this incertitude to influence the outcome of the inchoate propertization. Thus, unlike ownership of conventional, tangible properties (e.g., land or chattel), the successful (or, equally, unsuccessful) privatization of agricultural genetic sequences is dependent upon the process of creating the property, itself. Employing grounded theory methodologies, this dissertation analyzes five case studies to develop a unique model describing the uncertain process of creating property from agricultural genetics and facilitate explaining why certain propertization attempts are successful while others are not.

DNA assembly and rational design are cornerstones of synthetic biology. While many DNA assembly standards have been published in recent years, only the Modular Cloning standard, or MoClo, has the advantage of publicly available part libraries for use in plant, yeast, and mammalian systems. No multipart modular library has previously been developed for use in prokaryotes. Building upon the existing MoClo assembly framework, we developed a collection of DNA parts and optimized MoClo protocols for use in E. coli. We present this assembly standard and library along with part characterization, design strategies, potential applications, and troubleshooting. Developed as part of the Cross-disciplinary Integration of Design Automation Research (CIDAR) lab collection of tools, the CIDAR MoClo Library is publicly available and contains promoters, ribosomal binding sites, coding sequences, terminators, vectors, and a set of fluorescent control plasmids. Optimized protocols reduce reaction time and cost by >80% from previously published protocols. The CIDAR MoClo Library is the first bacterial DNA part library compatible with a multipart assembly standard. To demonstrate the utility of the CIDAR MoClo system in a traditional biology context, we used the library and previous expression data to create a series of dual expression plasmids. In this manner, we produced a dual expression plasmid capable of expressing equimolar amounts of two variants of rabbit aldolase, a His-tagged wildtype protein and a single-amino-acid substitution mutant deficient in binding actin. This expression plasmid will enable the production of dimer-of-dimer heterotetramers needed for structural determination of the actin-aldolase interaction by electron microscopy. To employ CIDAR MoClo in a synthetic biology context, we produced a bioelectronic pH-mediated genetic logic gate with DNA circuits built using MoClo and integrated with Raspberry Pi computers, Twitter, and 3D printed components. Logic gates are an increasingly common biological tool with applications in cellular memory and biological computation. MoClo facilitates rapid iteration of genetic designs, better enabling the development of cellular logic. The CIDAR MoClo Library and assembly standard enable rapid design-build-test cycles in E. coli making this system advantageous for use in many areas of synthetic biology as well as traditional biological research.

Made available in DSpace on 2014-10-09T12:41:47Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:07:18Z (GMT). No. of bitstreams: 0 / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Orientador: José Maria Ferreira Jardim da Silveira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Economia / Made available in DSpace on 2018-08-16T09:40:16Z (GMT). No. of bitstreams: 1 Borges_IzaiasdeCarvalho_D.pdf: 3979836 bytes, checksum: 7c9e18b48e3ecdadc3004f5b5e8dd5de (MD5) Previous issue date: 2010 / Resumo: Nos últimos 15 anos a engenharia genética vem sendo usada para o desenvolvimento de cultivos geneticamente modificados (GM) com diversos atributos de interesses para a produção agrícola, tais como tolerância a herbicidas, resistência a insetos e modificações nas características nutricionais de diversos cultivos. Os cultivos GM estão sendo utilizados na produção de produtos de grande importância no mercado mundial de commodities agrícolas, tais como algodão, milho e soja. Agricultores de diversos países, incluindo dos grandes produtores agrícolas mundiais, como Argentina, Brasil, Estados Unidos, China e Índia, estão sendo beneficiados com o uso de cultivos GM. Além dos benefícios econômicos, como a redução dos custos de produção e das perdas causadas pelos ataques de pragas, os cultivos GM estão apresentando também benefícios ambientais e sociais, ambos associados com a redução no uso de pesticidas. Mas a despeito da ampla aceitação pelos agricultores e dos benefícios ambientais e sociais observados, os cultivos GM estão enfrentando rejeição por uma parte da opinião pública e diversos governos estão adotando políticas regulatórias de restrições à produção, à importação e ao uso dos cultivos GM para a produção de alimentos. Assim, o objetivo desta tese foi entender as razões pelas quais tanto a opinião pública quanto os governos de diversos países estão se posicionando contra o uso dos cultivos GM na produção agrícola, a despeito dos benefícios observados pelos agricultores. Os estudos de opinião pública mostram que a rejeição aos cultivos GM é maior do que a rejeição as aplicações da engenharia genética em outras áreas, como por exemplo, na indústria farmacêutica. Esta diferença sugere que a rejeição aos cultivos GM está relacionada com as peculiaridades dos cultivos GM. A primeira é que na maioria dos casos eles são utilizados na produção de alimentos, um segmento no qual a variável segurança tem um grande peso nas decisões dos consumidores. A segunda característica é que a maior parte dos cultivos GM produzidos atualmente foram desenvolvidos para melhorar o processo produtivo, o que resulta em uma assimetria de percepção dos seus benefícios ao longo da cadeia produtiva, ou seja, os agricultores tendem a perceber mais os seus benefícios do que os consumidores. Uma terceira característica dos cultivos GM é que ao contrário das aplicações da engenharia genética na indústria farmacêutica, eles são expostos ao meio ambiente. Estas três características resultam em grande percepção de riscos, tanto para a saúde humana quanto para o meio ambiente, e baixa percepção dos benefícios. Entre os especialistas - público não leigo - também existem rejeição aos cultivos GM, embora em menor grau do que entre o público leigo. Um estudo empírico realizado no Brasil como 65 especialistas, incluindo pesquisadores de instituições públicas de pesquisa, professores universitários e profissionais de empresas privadas, mostrou que entre eles não só há discordância quanto aos benefícios e aos riscos dos cultivos GM, mas principalmente quanto aos tipos de riscos e de benefícios que devem ter maior peso nas decisões políticas com relação à estes cultivos / Abstract: Over the past 15 years genetic engineering has been used to develop genetically modified crops (GM) with several attributes of interest to agricultural production, such as herbicide tolerance, insect resistance and changes in nutritional characteristics of different crops. GM crops are being used to produce products of great importance in the agricultural global market, for commodities such as cotton, corn and soybeans. In several countries, including the world's major agricultural producers like Argentina, Brazil, United States, China and India, farmers are having the benefits of GM crops use. Besides economic benefits such as reduced production costs and reduced losses from pest attacks, GM crops are also presenting environmental and social benefits, both associated with a reduction in pesticide use. But despite the wide acceptance by farmers and the observed environmental and social benefits, GM crops are facing rejection by part of the public and many governments are adopting policies of regulatory restrictions on production, importation and the use of GM crops for food production. The purpose of this thesis was to understand the reasons why the public opinion and the governments of several countries are positioning themselves against the use of GM crops in agricultural production, despite the benefits observed by farmers. Studies of public opinion show that the rejection of GM crops is greater than the rejection of genetic engineering applications in other areas, such as the pharmaceutical industry. This difference suggests that the rejection of GM crops is related to their peculiarities. The first peculiarity of GM crops is that in most cases they are used in food production, a segment in which food safety weighs heavily on consumers' decisions. The second peculiarity is that most of the GM crops currently produced were made to improve the production process, resulting in an asymmetry in the perception of their benefits throughout the supply chain, ie, farmers tend to perceive more of such benefits than consumers. A third peculiarity of GM crops is that, because they are produced in extensive contact with nature, they arouse concerns about possible impacts on biodiversity. These three features result in increased awareness of risks for both human health and for the environment, and low perceived benefits. This study aimed to analyze the perception of risk and benefits of GM crops in Brazil. The methodology used was the application of questionnaires to experts. We used a multiple criteria method of hierarchical analysis for the preparation of the questionnaire. The results showed that among Brazilian specialists there is disagreement about the benefits and risks of GM crops, and especially regarding the types of risks and benefits that should have greater weight in policy decisions related to the use of GM crops in Brazilian agriculture / Doutorado / Teoria Economica / Doutor em Ciências Econômicas

Made available in DSpace on 2014-10-09T12:41:47Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:07:18Z (GMT). No. of bitstreams: 0 / Este trabalho utilizou pulsos laser ultracurtos para modificar, de forma controlada, as características dimensionais de nanopartículas de prata em solução aquosa. Para atingir este objetivo foram empregados algoritmos genéticos e circuitos microfluídicos. Utilizou-se um conformador temporal de pulsos ultracurtos para criar diversos perfis temporais de pulsos que irradiaram soluções de nanopartículas de prata. Estes perfis temporais foram ajustados em tempo real, visando otimizar o resultado do experimento, quantificada pela diminuição do diâmetro médio das nanopartículas nas soluções irradiadas. Uma vez que cada experimento de minimização do diâmetro das nanopartículas exigiu centenas de medidas, sua realização foi possível em decorrência da utilização de um circuito microfluídico construído especialmente para este trabalho. Neste circuito é possível utilizar pequenas quantidades de amostra, levando a curtos tempos de irradiação e medição, além da evidente economia de amostras. Para a realização deste trabalho foi elaborado e testado um algoritmo genético interfaceado a diversos equipamentos, incluindo um filtro acustóptico dispersivo programável que modifica as características temporais dos pulsos ultracurtos, através da introdução de componentes de fases espectrais nestes pulsos. Utilizando o algoritmo genético e o filtro acustóptico dispersivo programável foram realizados experimentos de encurtamento da duração temporal dos pulsos ultracurtos provenientes do sistema laser, resultando na obtenção de pulsos com durações próximas às limitadas por transformada de Fourier. Além disso, foram realizados experimentos para a otimização do processo evolutivo do algoritmo genético escrito em Labview. Os experimentos de irradiação de soluções de nanopartículas de prata mostraram que, ao conformar a duração dos pulsos utilizados nas irradiações, pôde-se controlar as dimensões destas nanopartículas, diminuindo seu tamanho médio por um fator 2. Esses experimentos caracterizam a irradiação de nanopartículas por lasers de pulsos ultracurtos como uma importante técnica de controle de características de nanopartículas. / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Orientadores: Telma Teixeira Franco, Ana Carolina Deckmann / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-25T22:35:19Z (GMT). No. of bitstreams: 1 Coradini_AlessandroLuisVenega_M.pdf: 2904811 bytes, checksum: 79be0b1291d380e8c86253645ed455d8 (MD5) Previous issue date: 2014 / Resumo: A crise energética mundial aliada aos problemas ambientais tem despertado grande preocupação mundial, aumentando assim a busca por combustíveis mais "limpos" que possam substituir os já existentes. Dentre estes substitutos podemos destacar os chamados biocombustíveis, como o etanol e o biodiesel. No caso da produção de biodiesel, diversas fontes de lipídios são consideradas, incluindo óleos vegetais, gorduras animais e óleos reciclados. O uso de microrganismos oleaginosos foi apresentado como uma fonte alternativa de óleos e gorduras de baixo custo (Meng et al., 2009). O óleo produzido por microrganismos pode ter como substrato qualquer fonte de carbono, incluindo bagaços e resíduos agrícolas em geral, evitando tanto a competição por áreas de plantio como agregando valor a dejetos de outros setores agrícolas. A esta abordagem dá-se o nome de "biocombustíveis de segunda geração". Neste contexto, as leveduras surgem como fortes candidatas à produção de biodiesel, uma vez que diversas ferramentas de cultivo e manipulação já foram implantadas especialmente para o uso de leveduras em processos industriais e biotecnológicos. Entretanto, nem todas as espécies de leveduras são adequadas para a produção de óleo, uma vez que a capacidade de acumular grandes quantidades de lipídeos varia significativamente entre as espécies. As leveduras do gênero Saccharomyces, por exemplo, apresentam de 8-15 de lipídeos totais em relação à massa seca. Outras, como as espécies Rhodotorula graminis, Rhodotorula gracilis e Lipomyces starkeyi, apresentam cerca de 30%, 60% e 65% de lipídeos totais, respectivamente. Entretanto, o emprego de microrganismos selvagens (i.e., não domesticados) limita o controle dos parâmetros que afetam o processo produtivo, uma vez que as bioconversões de interesse dependem de ajustes metabólicos desconhecidos, e a falta de conhecimento genético destes microrganismos dificulta a aplicação de técnicas de manipulação genética. Assim, o presente estudo teve como objetivo "domesticar" a linhagem DSM70826 da levedura Lipomyces starkeyi tornando-a de fácil manipulação genética e tornando-a uma plataforma para posterior introdução de características que possam melhorar o seu desempenho quanto ao acúmulo de lipídeos para produção de biodiesel. Uma vez que foi realizado o sequenciamento do genoma desta levedura durante trabalho anterior conduzido em nosso laboratório, também objetivamos utilizar este banco de dados para identificar e estudar alguns genes considerados relevantes aos processos fermentativos de xilose e também ao acúmulo de lipídeos, auxiliando a elucidar as principais rotas metabólicas envolvidas nestes processos / Abstract: The global energy crisis combined with environmental problems has aroused great worldwide concern, increasing the search for "cleaner" fuels that can replace existing ones. Among these substitutes the so-called biofuels, such as ethanol and biodiesel can be highlighted. In the case of biodiesel, different fat sources are considered, including vegetable oils, animal fats and recycled oils. The use of oleaginous microorganisms was presented as an alternative source of low cost oils and fat (Meng et al., 2009). The oil produced by microorganisms can use any substrate as carbon source, including bagasse and agricultural waste in general, avoiding the competition for growing areas and adding value to waste of other agricultural sectors. This approach is called "second generation biofuels". In this context, yeasts appear as strong candidates for biodiesel production, since many manipulation and cultivation tools have been implemented especially for the use of yeast in industrial and biotechnological processes. However, not all species of yeasts are suitable for the production of oil, since the ability to accumulate large amounts of lipids varies significantly between species. Yeasts of the genus Saccharomyces, for example, show 8-15% of total lipids in the dry mass. Others, such as the species Rhodotorula graminis, Rhodotorula gracilis and Lipomyces starkeyi, have about 30 %, 60% and 65% of total lipids, respectively. However, the use of wild microorganisms (i.e., non-domesticated) limits the control parameters that affect the production process, since bioconversions of interest depend of unknown metabolic adjustments, and lack of genetic knowledge of these microorganisms hinders the application of genetic manipulation techniques. Therefore, the present study aimed to "domesticate" the yeast strain Lipomyces starkeyi DSM70826 making it easy genetic manipulation and making it a platform for subsequent introduction of features that can improve your performance on lipid accumulation for production of biodiesel. Since the genome sequencing of this yeast was performed during previous work conducted in our laboratory, we also aim to use this database to identify and study some genes considered relevant to xylose fermentation processes and also to lipid accumulation, helping to elucidate the main metabolic pathways involved in these processes / Mestrado / Engenharia de Processos / Mestre em Engenharia Química

Ph.D. (Biochemistry) / Genetically modified (GM) crops were first introduced in the 1980s for the production of medicinal products. Since then, areas designated to GM crops have expanded drastically, with the GM crops grown to enhance agricultural productivity, improve agricultural practices, and as a tool to address potential pressures that will be faced by the agricultural sector and to address the issue of food security. Currently, cultivated GM crops include cotton, maize, rapeseed and soybean, carrying agronomic traits such as herbicide tolerance and insect resistance. Following the genetic modification of crops, three possible outcomes can be anticipated: these outcomes include the GM crop produced being equivalent to its untransformed counterpart, the GM crop differing from its untransformed counterpart with several well-defined characteristics, and the GM crop differing from its untransformed counterpart with a multitude of complex characteristics. In cases where the GM crop is equivalent to the untransformed counterpart, no further testing is needed. In instances where several well-defined and characterised differences are found between the GM crop and the untransformed counterpart, safety assessments are performed targeting these differences. The assessments will determine the impact of these unintended and unexpected alterations of the intended enhancement of the GM crops. However, methods currently used to assess GM crops have been found to be lacking, since they only focus on environmental and product-specific risks. Further evidence is essential, as part of GM crop safety assessment, on the molecular characterisation of these crops. This evidence is based on the potential impact of the transformation event, integration of the transgene into the host plant, as well as unintended alterations such as altered gene expression that may occur to the host plant. These events may assist in the further detection of potential dangers of the GM crop. As a result of these highlighted gaps, a project was formulated to study the unintended genomic alterations that may occur during and following the production of a transgenic plant...

Abstract Background – The heart cell is a prime example of a system, in which numerous interconnected regulatory mechanisms affect the dynamic balance of cellular function. The function of the system emerges from the interactions of its components rather than from their individual properties. Thus, it is a challenging task to understand the causal relations within such a system, based on the analysis of experimental results. Facing this complexity, the systems biological approach has gained interest during recent years, since with using it we can make an effort to observe, quantitatively and simultaneously, multiple components and their interdependencies in biological networks. Methods and aims – One of the most important tools in systems biology is mathematical modelling. In this thesis, novel model components have been developed and existing components integrated to describe mathematically the calcium dynamics in cardiac myocytes with improved physiological accuracy. Special attention was paid to both the activity-dependent and automatic regulation of the dynamics. This enabled the quantitative analysis of the regulation’s role in both physiological and pathophysiological conditions. Results – Validation of the novel model components that describe the calcium transport mechanisms indicates that the developed schemes are accurate and applicable also beyond the normal physiological state of the cardiac myocyte. Results also highlight the importance of autoregulation of calcium dynamics in the excitation-contraction coupling. Furthermore, the analysis indicates that the CaMK-dependent regulation of the calcium uptake to and release from the sarcoplasmic reticulum calcium stores could have substantial roles as downstream effectors in beta-adrenergic stimulation. Conclusions – Results emphasize mathematical modelling as a valuable complement to experiments in understanding causal relations within complex biological systems such as the cardiac myocytes. That is, rigorous data integration with mathematical models can provide significant insight to the quantitative role of both the individual model components and the interconnected regulatory loops. This is especially true for the analysis of genetically engineered animal models, in which the intended modification is always accompanied by compensatory changes that can mask to a varying degree the actual phenomenon of interest.

Please read the abstract in the section 00front of this document / Dissertation (MSc (Microbiology))--University of Pretoria, 2005. / Microbiology and Plant Pathology / unrestricted