Conforme novas tecnologias e metodologias estão surgindo, e pesquisadores estão sedentos por ferramentas moleculares mas rápidas, mais eficientes e fáceis de usar, dominar os princípios e tecnologias do design de vetores e padronização de partes biológicas tornaram-se desafios fundamentais. Isso está abrindo espaço para o surgimento de uma disciplina inteiramente nova chamada Biologia Sintética. Esta área de estudo inovadora combina partes e módulos biológicos para criar sistemas mais confiáveis e robustos. Linhagens fúngicas são comumente alvo desses estudos, não apenas porque muitos achados fundamentais em relação à clonagem molecular surgiram das lições dadas por elas, mas também devido a um imenso e inexplorado potencial desses organismos em uma ampla gama de aplicações - desde biocombustíveis e produção de químicos finos até terapias biomédicas. Neste contexto, a presente dissertação é dividida em duas partes: a primeira diz respeito ao design e construção de uma ferramenta modular e versátil para ser aplicada em várias linhagens de fungos. Essa ferramenta é um plasmídeo binário para transformação mediada pro Agrobacterium tumefaciens, que foi construído em quatro diferentes versões contendo GFP ou mCherry como proteínas repórter e um gene sintético de resistência à higromicina como marcador de seleção. O vetor foi validado em Paracoccidioides lutzii, um patógeno oportunista humano dimórfico que é muito importante para medicina, mas ainda carecia de ferramentas genéticas eficientes. A segunda parte consiste na criação de uma biblioteca de promotores para a levedura oleaginosa Rhodosporidium toruloides, um promissor hospedeiro para a produção de bioprodutos a partir de biomassa, uma vez que pode eficientemente consumir açúcares C5 e C6 e aromáticos derivados da lignina. Vinte e nove promotores foram testados em um cassete de duplo-repórter - compreendendo ambas as proteínas fluorescentes GFP e mRuby - utilizando citometria de fluxo para análise de células únicas. A coleção de promotores apresentados neste trabalho é a maior disponível para R. toruloides até o momento e foi um avanço indispensável para superar a10 escassez de ferramentas para este organismo. Notavelmente, também apresentamos os primeiros promotores bidirecionais descritos para essa levedura e otimizamos o protocolo de transformação. Portanto, a Biologia Sintética foi eficientemente aplicada para expandir a coleção de partes biológicas padronizadas e otimizar vetores para transformação e manipulação genética de fungos. Estas ferramentas são de valor imediato e são aplicáveis a desafios muito distintos, mas igualmente importantes: a busca de novas soluções para a saúde humana e para uma economia bio-sustentável. / As new technologies and methodologies are surfacing, and researchers are now eager for fast, enhanced and easy-to-use molecular tools, mastering the principles and technologies of vector design and standardization of biological parts have become fundamental challenges. This is making room for the rise of an entirely novel discipline called Synthetic Biology. This innovative field of study combines biological parts and modules to create more reliable and robust systems. Fungal strains are commonly the target of these studies, not only because several fundamental findings regarding molecular cloning arose from lessons given by them, but also due to an immense and much unexplored potential of those organisms in a wide range of applications - ranging from biofuels and fine chemicals production to biomedical therapies. In this context, the present dissertation is divided in two parts: the first one concerns the design and construction of a modular and versatile tool to be applied in several fungal strains. This tool is a plasmid binary vector for Agrobacterium tumefaciens-mediated transformation, which was built in four different versions containing either GFP or mCherry as reporter proteins and a synthetic hygromycin resistance gene as selection marker. The vector was validated in Paracoccidioides lutzii, a dimorphic human opportunist pathogen that is very important for health care but was still lacking efficient genetic tools. The second part consists in the creation of a promoter library for the oleaginous yeast Rhodosporidium toruloides, a promising host for the production of bioproducts from biomass since it can efficiently consume C5 and C6 sugars and lignin-derived aromatics. Twenty-nine promoters were tested with a dual-reporter cassette - comprising both GFP and mRuby fluorescent proteins - using flow cytometer for single-cell analysis. The assortment of promoters presented in this work is the largest set available for R. toruloides until now and was an imperative advancement to overcome the scarcity of tools for this organism. Remarkably, we also presented the first bidirectional promoters described for this yeast and optimized the transformation protocol. Thus, we efficiently applied Synthetic Biology to expand the collection of standard biological parts and to12 optimize vectors for fungal transformation and genetic manipulation. These tools are of immediate value and are applicable for very distinct but equally important challenges: the pursuit of new solutions for human health and for a sustainable biobased economy

O emprego de novas tecnologias, como a transgenia, associadas ao melhoramento convencional de cana-de-açúcar apresenta grande potencial no combate a pragas e desenvolvimento de novas variedades. Entretanto, a falta de especificidade na expressão temporal e/ou local dos genes introduzidos tem se mostrado um fator limitante para o sucesso de produtos derivados da transgenia. Os promotores das metalotioneínas, por exemplo, podem representar uma alternativa ao uso de promotores constitutivos, particularmente, aqueles de metalotioneínas do tipo 1 (MT1) por apresentarem níveis elevados de expressão, em diferentes tecidos/órgãos e serem responsivos a estresses bióticos e abióticos. O uso de promotores sintéticos, contendo apenas elementos-cis, como GCG-like, W boxes e JERE, tem sido relevante por induzirem a expressão gênica local em resposta ao ataque de agentes bióticos. Este trabalho teve por objetivo a caracterização funcional de promotores de genes de metalotioneínas de cana-de-açúcar e o uso de elementos regulatórios sintéticos e, em paralelo, avaliou-se o uso de promotores sintéticos no controle da expressão gênica em cana-de-açúcar. Após as análises de expressão transiente, verificou-se que o promotor SoMT1b apresentou atividade GUS e GFP em epitélios de cebola e os promotores sintéticos, 4X Wbox, 4X GCC-like, 4X JERE e 4xW 4xS-box, e o promotor SoMT1b foram capazes de dirigir a expressão do gene repórter uidA (GUS) em calos embriogênicos de cana-de-açúcar. Em análise de expressão estável, o promotor SoMT1b foi capaz de dirigir a expressão do gene GUS para os frutos e sementes de tomate \'Micro-Tom\', mas não foi responsivo a estresse por herbivoria, cádmio e cobre. Também foi realizada a transformação de plantas de cana-de-açúcar, as quais ainda estão sendo analisadas. Os resultados obtidos demonstram a funcionalidade do promotor SoMT1b / New technologies, like genetic transformation, associated with conventional breeding of sugarcane have a large potential in developing new varieties tolerant to biotic and abiotic stress. However, the lack of specificity in the spatial and/or temporal expression of the introduced genes has been a limited factor for the success of products derived from transgeny. Metallothionein promoters, for instance, can represent an alternative to the use of constitutive promoters, particularly those from metallothionein type 1 (MT1) because they present high expression levels in different tissues / organs and are responsive to biotic and abiotic stress. Another alternative is the use of synthetic promoters which contain only cis elements, as GCG-like, W-box and JERE, which induces local gene expression in response to pathogen attack. In this work, we aimed to make the functional characterization of sugarcane metallothionein promoters and synthetic regulatory elements, in parallel, we evaluated the use of synthetic promoters in the control of gene expression in sugarcane. After transient expression analyzis, it was found that SoMT1b promoter was able to control the expression of the reporter genes to GUS and GFP in onion epithelium and GUS in sugarcane embryogenic calli. Additionally, synthetic promoters 4X Wbox, 4X GCC-like, 4X JERE and 4xW 4xS-box were able to direct the expression of the gene uidA (GUS) in embryogenic calli of sugarcane. In stable transformation analysis, SoMT1b promoter was capable of directing uidA expression in fruits and seeds of tomato cv. \'Micro-Tom\', but it was not responsive to herbivory, cadmium and copper stress. It was also carried out the transformation of sugarcane plants with the construction containing the SoMT1b promoter, but these are still being analyzed. The results demonstrate the functionality of the SoMT1b promoter in sugarcane and tomato

O emprego de novas tecnologias, como a transgenia, associadas ao melhoramento convencional de cana-de-açúcar apresenta grande potencial no combate a pragas e desenvolvimento de novas variedades. Entretanto, a falta de especificidade na expressão temporal e/ou local dos genes introduzidos tem se mostrado um fator limitante para o sucesso de produtos derivados da transgenia. Os promotores das metalotioneínas, por exemplo, podem representar uma alternativa ao uso de promotores constitutivos, particularmente, aqueles de metalotioneínas do tipo 1 (MT1) por apresentarem níveis elevados de expressão, em diferentes tecidos/órgãos e serem responsivos a estresses bióticos e abióticos. O uso de promotores sintéticos, contendo apenas elementos-cis, como GCG-like, W boxes e JERE, tem sido relevante por induzirem a expressão gênica local em resposta ao ataque de agentes bióticos. Este trabalho teve por objetivo a caracterização funcional de promotores de genes de metalotioneínas de cana-de-açúcar e o uso de elementos regulatórios sintéticos e, em paralelo, avaliou-se o uso de promotores sintéticos no controle da expressão gênica em cana-de-açúcar. Após as análises de expressão transiente, verificou-se que o promotor SoMT1b apresentou atividade GUS e GFP em epitélios de cebola e os promotores sintéticos, 4X Wbox, 4X GCC-like, 4X JERE e 4xW 4xS-box, e o promotor SoMT1b foram capazes de dirigir a expressão do gene repórter uidA (GUS) em calos embriogênicos de cana-de-açúcar. Em análise de expressão estável, o promotor SoMT1b foi capaz de dirigir a expressão do gene GUS para os frutos e sementes de tomate \'Micro-Tom\', mas não foi responsivo a estresse por herbivoria, cádmio e cobre. Também foi realizada a transformação de plantas de cana-de-açúcar, as quais ainda estão sendo analisadas. Os resultados obtidos demonstram a funcionalidade do promotor SoMT1b / New technologies, like genetic transformation, associated with conventional breeding of sugarcane have a large potential in developing new varieties tolerant to biotic and abiotic stress. However, the lack of specificity in the spatial and/or temporal expression of the introduced genes has been a limited factor for the success of products derived from transgeny. Metallothionein promoters, for instance, can represent an alternative to the use of constitutive promoters, particularly those from metallothionein type 1 (MT1) because they present high expression levels in different tissues / organs and are responsive to biotic and abiotic stress. Another alternative is the use of synthetic promoters which contain only cis elements, as GCG-like, W-box and JERE, which induces local gene expression in response to pathogen attack. In this work, we aimed to make the functional characterization of sugarcane metallothionein promoters and synthetic regulatory elements, in parallel, we evaluated the use of synthetic promoters in the control of gene expression in sugarcane. After transient expression analyzis, it was found that SoMT1b promoter was able to control the expression of the reporter genes to GUS and GFP in onion epithelium and GUS in sugarcane embryogenic calli. Additionally, synthetic promoters 4X Wbox, 4X GCC-like, 4X JERE and 4xW 4xS-box were able to direct the expression of the gene uidA (GUS) in embryogenic calli of sugarcane. In stable transformation analysis, SoMT1b promoter was capable of directing uidA expression in fruits and seeds of tomato cv. \'Micro-Tom\', but it was not responsive to herbivory, cadmium and copper stress. It was also carried out the transformation of sugarcane plants with the construction containing the SoMT1b promoter, but these are still being analyzed. The results demonstrate the functionality of the SoMT1b promoter in sugarcane and tomato

Thesis (PhD (Genetics. Plant Biotechnology))--University of Stellenbosch, 2006. / The young internodes of sugarcane are ideal targets for altering metabolism, through genetic manipulation, to potentially control known fungal diseases such as Smut or to increase sucrose yields in these regions that are currently being discarded. At present, no regulatory sequences that specifically drive transgene expression in young developing sugarcane tissues are available. The objective of this study was therefore to isolate and evaluate such a sequence. The promoter targeted for isolation in this study regulates the expression of UDP-glucose dehydrogenase (EC 1.1.1.22), an enzyme which catalyses the oxidation of UDP-glucose to UDP-glucuronic acid, a precursor for structural polysaccharides which are incorporated into the developing cell wall. A strong correlation between the expression of UDP-glucose dehydrogenase and a demand for structural polysaccharides in developing tissues could therefore be expected. The first part of this study addressed the general practicality of promoter isolation from sugarcane, a complex polyploid. A gene encoding UDP-glucose dehydrogenase was isolated from a sugarcane genomic library. The gene contains an open reading frame (ORF) of 1443 bp, encoding 480 amino acids and one large intron (973 bp), located in the 5’-UTR. The derived amino acid sequence showed 88 – 98% identity with UDP-glucose dehydrogenase from other plant species, and contained highly conserved amino acid motifs required for cofactor binding and catalytic activity. Southern blot analysis indicates a low copy number for UDP-glucose dehydrogenase in sugarcane. The possible expression of multiple gene copies or alleles of this gene was investigated through comparison of sequences amplified from cDNA prepared from different tissues. Although five Single Nucleotide Polymorphisms (SNP) and one small-scale insertion/deletion (INDEL) were identified in the aligned sequences, hundred percent identity of the derived amino acid sequences suggested the expression of different alleles of the same gene rather than expression of multiple copies. The finding that multiple alleles are expressed to provide the required level of a specific enzyme, rather than the increased expression of one dominant allele, is encouraging for sugarcane gene and promoter isolation. In the second part of the study the suitability of UDP-glucose dehydrogenase as a target for the isolation of a developmentally regulated promoter was investigated. The contribution of UDP glucose dehydrogenase to pentan synthesis, as well as the expression pattern and subcellular localisation of the enzyme in mature sugarcane plants was studied at the tissue and cellular level. Radiolabelling with positionally labelled glucose was used to investigate the relative contributions of glycolysis, the oxidative pentose phosphate pathway and pentan synthesis to glucose catabolism. Significantly (P=0.05) more radiolabel was released as CO2 from [6-14C]- glucose than [1-14C]-glucose in younger internodes 3, 4 and 5, demonstrating a significant contribution of UDP-glucose dehydrogenase to glucose oxidation in the younger internodes. In addition, there was significantly (P=0.05) more radiolabel in the cell wall (fiber) component when the tissue was labelled with [1-14C]-glucose rather than [6-14C]-glucose. This also demonstrates a selective decarboxylation of glucose in position 6 prior to incorporation into the cell wall and is consistent with a major role for UDP-glucose dehydrogenase in cell wall synthesis in the younger internodes. Expression analysis showed high levels of expression of both the UDP-glucose dehydrogenase transcript and protein in the leafroll, roots and young internodes. In situ hybridisation showed that the UDP-glucose dehydrogenase transcript is present in virtually all cell types in the sugarcane internode, while immunolocalisation showed that the abundance of the protein declined in all cell types as maturity increased. Results obtained confirmed that this enzyme plays an important role in the provision of hemicellulose precursors in most developing tissues of the sugarcane plant, indicating that UDP-glucose dehydrogenase was indeed a suitable target for promoter isolation. Lastly, the promoter region and first intron, located in the 5’-untranslated region (UTR) of this gene, were isolated and subsequently fused to the GUS reporter gene for transient expression analysis and plant transformation. Transient expression analysis showed that the presence of the intron was essential for strong GUS expression. Analysis of stably transformed transgenic sugarcane plants, evaluated in a green house trial, showed that the isolated promoter is able to drive GUS expression in a tissue specific manner under these conditions.

Dissertation (PhD)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: To manipulate recombinant microorganisms for industrial processes, controllable genetic systems are needed that can coordinate expression of recombinant metabolic pathways. All components are sensitive to change and thus putative targets for modification and genetic elements and regulatory systems need to be understood and determined. Central in gene regulation is the transcription activators that mediate gene transcription mechanisms by binding to promoters in response to environmental signals. Promoter engineering entails the modification of transcription factors and their target promoters. In this study, a metabolic control system in Saccharomyces cerevisiae was constructed that would allow induction in response to physiological environment, specifically hypoxia and low temperature conditions. Two approaches were undertaken to find such a system. Firstly, a bi-directional reporter gene cloning vector was designed to search for novel hypoxiainducible promoters. Secondly, a transcription regulatory circuit was built, consisting of an inducible transcription regulator and promoter with a reporter gene through which it mediates transcription. Advantage was taken of the modular nature of proteins and functional domains originating from different transcriptional proteins were combined. A search for promoter elements sensitive to hypoxia from a S. cerevisiae genomic DNA (gDNA) library, using a bi-directional cloning vector, did not yield highly inducible promoters. It was concluded that a multitude of signals overlap, rendering genetic induction difficult to control. A synthetic regulatory system would minimize the impact of these multiple interactions. Such a genetic circuit was constructed, consisting of a chimeric transcription activator and a target fusion promoter. The chimeric transcription activator consisted of the GAL4 DNA binding domain, ADR1 TADIII transactivation domain and three domains of the MGA2 regulatory protein. The functional domains of Mga2p responsible for unregulated expression (at high basal levels) under both aerobic and hypoxia conditions were located, as well as a further upregulation under low temperature, and were mapped to the Nterminal and mid-Mga2p regions. A target fusion promoter consisting of a partial GAL10/1 promoter sequence and a Trichoderma reesei core xyn2 promoter were constructed as target for this chimeric transactivator. This synthetic promoter was fused to the T. reesei xyn2 open reading frame encoding for a readily assayable β-xylanase activity. Both the chimeric transactivator and fusion promoter-reporter gene cassettes were expressed from the same episomal plasmid, named pAR. Transformed into S. cerevisiae Y294, this regulatory system induced transcription under aerobic and hypoxia conditions. Furthermore, the reporter gene expression was upregulated by the chimeric transactivator at low temperatures. The chimeric transactivator mediated a seven-fold induction of the reporter gene under aerobic conditions in S. cerevisiae Y294 when transformed with plasmid AR. A two- to three-fold induction at 23ºC was reported under anaerobic conditions, relative to a reference strain expressing a transcription activator without the Mga2p domains. At 30ºC, a two- to three-fold induction under aerobic conditions and similar induction under oxygen-limited conditions were observed. Replacing the reporter gene with your favorite gene (for example a recombinant enzyme) and incorporating such a pAR system into a recombinant yeast should induce expression of the chosen gene under low temperatures, both aerobic and anaerobically (thus creating a controllable system). The system also has wider application in identifying other transcription factors’ signal-sensitive domains. The design of this system provides the ability to add a linker to a transactivator and to either create specific signal sensitivity or relieve the regulator of its signal dependence. It creates an easy system for assessing other transactivators and their domains with unknown functions and thus provides a ”workhorse and prospector in one”. / AFRIKAANSE OPSOMMING: Vir die manipulering van rekombinante mikroörganismes vir industriële prosesse word beheerbare genetiese stelsels benodig om gekoördineerde uitdrukking van rekombinante metaboliese weë teweeg te bring. Alle komponente van sulke stelsels is sensitief vir verandering en genetiese elemente en reguleerbare sisteme moet dus deeglik verstaan of bepaal word. Sentraal tot geenregulering is die transkripsie-aktiveerders wat geentranskripsie beheer deur aan promoters te bind in reaksie op eksterne omgewingsfaktore. Promotoringenieurswese behels wysigings van transkripsiefaktore en hul teikenpromotors. In hierdie studie is 'n genetiese beheerstelsel vir Saccaromyces cerevisiae ontwikkel wat induksie in reaksie tot spesifieke fisiologiese omgewingreaksies, naamlik hipoksie- en lae temperatuur, toelaat. Twee benaderings is gevolg: eerstens is ‘n tweerigting verklikker-geen vektor ontwikkel en gebruik om vir unieke induseerbare hipoksie-promoters te soek. Tweedens is ‘n transkripsie reguleringstelsel gebou wat uit ‘n induseerbare transkripsiereguleerder and promotor met ‘n verklikkergeen bestaan, waardeur transkripsie bemiddel kan word. Hierdie benadering benut die modulêre onderbou van proteïene en funksionele domeine afkomstig vanaf verskillende transkripsiefaktore is gekombineer. 'n Soektog na hipoksie-sensitiewe promotors vanuit 'n Saccharomyces cerevisiae-genoom- DNA (gDNA), deur van ‘n tweerigting verklikker-vektor gebruik te maak, het ongelukkig nie hoogs-induseerbare promotors opgelewer nie. Die gevolgtrekking was dat ‘n veelvoud van seine met mekaar oorvleuel en die beheer van genetiese induksie dus bemoeilik. Die ontwikkeling van ‘n sintetiese regulering-sisteem kan die impak van die veelvuldige interaksies verminder. Vir dié doel is ‘n sintetiese reguleringstelsel ontwerp, bestaande uit ‘n chimeriese transkripsie-aktiveerder met ‘n teiken fusie-promotor. Die chimeriese transaktiveerder bestaan uit die GAL4 DNA bindingsdomein, die ADR1 TAD III transaktiveringsdomein en drie domeine van die Mga2 reguleringsproteïen. In die studie is die funksionele domeins van Mga2p betrokke by lae temperatuur-respons en ongereguleerde uitdrukking (teen hoë basale vlakke) onder beide aërobiese en anaërobiese toestande aangedui en is tot die N-terminaal en middel-Mga2p areas gekarteer. ‘n Teiken-fusie-promoter, bestaande uit 'n gedeeltelike GAL1/10 DNA promotoropeenvolging en ‘n Trichoderma reesei kern xyn2-promoter, is as teiken vir hierdie chimeriese transaktiveerder saamgestel. Hierdie sintetiese promotor is aan die T. reesei xyn2 oopleesraam, wat vir ‘n maklik meetbare β-xylanase aktiwiteit kodeer, gekoppel. Beide die chimeriese transaktiveerder and fusie-promoter-verklikker-geenkaset word vanaf dieselfde episomale plasmied, bekend as pAR, uitgedruk. Hierdie reguleringsisteem induseer transkripsie onder aërobiese en hipoksie toestande in S. cerevisiae Y294. Verder word die verklikkergeen se uitdrukking deur die chimeriese transaktiveerder by lae temperature verhoog. Die chimeriese transaktiveerder induseer ‘n sewe-voudige induksie van die verklikkergeen onder aërobiese toestande by 23ºC vanaf die pAR-stelsel in S. cerevisiae Y294. ‘n Twee- tot drie-voudige induksie teen 23ºC is onder hipoksie toestande gevind, relatief tot induksievlakke van ‘n verwysingstam met ‘n transaktiveerder sonder die Mga2 domeine. By 30ºC is ‘n twee- tot drie-voudige induksie onder aërobiese en lae suurstofvlakke waargeneem. Deur die verklikker geen met ‘n jou-gunsteling-geen te vervang (bv. ‘n rekombinante ensiem) en so 'n pAR-sisteem in ‘n rekombinante gis te inkorporeer, word uitdrukking onder lae temperature onder beide aërobiese- en anaërobiese toestande geïnduseer (en sodoende word ‘n reguleerbare sisteem geskep). Die sisteem het wyer toepassing om sein-sensitiewe domeine van ander transkripsiefaktore te identifiseer. Die ontwerp van die stelsel maak dit moontlik om 'n skakel tot die transaktiveerder by te voeg wat óf sensitiwiteit tot 'n spesifieke sein skep, óf die reguleerder vanaf seinafhanklikheid verlos. So word ‘n bruikbare stelsel vir die bestudering van ander transaktivators en hul domeine met onbekende funksie geskep – ‘n “werksesel en prospekteerder in een”.

Dissertation (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Understanding the complex nature of grapevine molecular biology is of great importance for viticulturists. Progress in the elucidation of key events on a genetic level could provide further insight into the underlying cues responsible for the precise control of physiological and metabolic changes during a specific condition such as fruit development. The use and analysis of molecular ‘tools’, such as promoters controlling the site and level of gene activity, could assist in the understanding of grapevine biology and serve as a platform for the future design and development of recombinant DNA protocols and strategies for Vitis vinifera L. A high-throughput gene expression system, cDNA-AFLPs, was successfully used to analyse large-scale transcriptional activity during berry ripening. Candidate cDNA fragments were selected on the basis of desired expression patterns and/or known gene function for subsequent promoter isolation. From three candidate cDNAs selected, the promoter of a gene encoding vacuolar pyrophosphatase (V-PPase) was isolated for computational and comparative analyses. Promoter activity was evaluated on a transient level using the green fluorescent protein (GFP) reporter gene. Comparative integration has allowed for putative correlation of cis-elements, acting as receptors within promoter regions, to regulate V-PPase gene expression in response to development, environmental stress and tissue-specificity. In this study, integration of genetic data have advanced the understanding and transcriptional role of a key enzyme (V-PPase) during grape ripening. Although never a replacement for experimental verification, this integrative strategy of combining gene expression profiles with bioinformatics and regulatory data will greatly assist in further elucidation of various other key components and regulatory cues associated with grapevine molecular biology. This study has allowed us to use molecular tools that could assist in gaining further insight into genetic complexities and could serve as a platform for a more refined genetic manipulation strategy in Vitis vinifera L. / AFRIKAANSE OPSOMMING: Begrip van die komplekse aard van wingerd molekulêre biologie is van groot belang vir wingerdkundiges. Vooruitgang in die begrip van belangrike gebeurtenisse op ń genetiese vlak behoort verdere insig in die onderliggende instruksies vir die noukeurige beheer van fisiologiese en metaboliese veranderinge tydens ń spesifieke kondisie soos vrug rypwording te bevorder. Die gebruik en analise van molekulêre ‘instrumente’ soos promoters, wat die posisie en vlak van geen aktiwiteit beheer, kan bydra tot n beter begrip van wingerd biologie en sodoende dien as ń platform vir die toekomstige ontwerp en ontwikkeling van rekombinante DNS (deoksiribonukleiensuur) protokolle en strategieë vir Vitis vinifera L. ń Hoë-kapasiteit geen uitdrukkings sisteem, nl. kDNS-AFLPs (komplementêre deoksiribonukleiensuur- geamplifiseerde fragment lengte polimorfisme), is suksesvol gebruik vir die analise van grootskaalse transkripsionele aktiwiteit tydens druif rypwording. Kandidaat kDNS fragmente is geselekteer, gebaseer op verlangde uitdrukkings-patrone en/of bekende geen funksie vir daaropvolgende promoter isolering. Van drie geselekteerde kandidaat kDNS fragmente, is die promoter van ń geen wat vakuolêre pirofosfatase (V-PPase) kodeer geïsoleer vir rekenaar- en vergelykende analise. Promoter aktiwiteit is op ń nie-stabiele vlak deur die gebruik van ń groen-fluoresserende proteien (GFP) verklikker geen geëvalueer. Vergelykende integrering het dit moontlik gemaak om veronderstelde korrelasies van cis-elemente, wat as reseptore binne ń promoter area dien, en die regulering van V-PPase geen uitdrukking, in reaksie tot ontwikkeling, omgewings stres en weefsel-spesifisiteit, te maak. Tydens hierdie studie, het die integrering van genetiese data gehelp om die transkripsionele rol van ń belangrike ensiem (V-PPase) tydens druif rypwording beter te verstaan. Alhoewel dit nooit ń plaasvervanger vir eksperimentele bewyse sal wees nie, kan hierdie gëintegreerde strategie, wat die kombinasie van geen-uitdrukkingsprofiele met bioinformatika en regulatoriese data behels, grootliks bydra om verskeie ander belangrike komponente en regulatorieseaanwysings geassosieërd met wingerd molekulêre biologie te ontrafel. Hierdie studie het verdere insig in genetiese kompleksiteite verleen, en kan nou dien as ń platform vir ń meer presiese genetiese manipulering strategie in Vitis vinifera L.

La régulation de l’apoptose est importante dans le maintient de l’homéostasie cellulaire et l’intégrité du matériel génétique. L’apoptose est un mécanisme cellulaire qui élimine les cellules endommagées. Le bon fonctionnement de cette voie biologique est crucial pour contrer la propagation des cellules avec leurs anomalies génétiques. La dérégulation des gènes codants pour des composantes de la voie intrinsèque de l’apoptose est fréquemment observée chez divers types de cancers, incluant la leucémie. Nous proposons que des polymor¬phis¬mes fonctionnels localisés dans la région régulatrice (rSNP) des gènes impliqués dans la voie d’apoptose intrinsèque auraient un impact significatif dans l’oncogenèse en modifiant le taux d’expression de ces gènes. Dans cette étude, nous avons validé, à l’aide d’une combinaison d’approches in silico et in vitro, l’impact fonctionnel de la variabilité génétique sous la forme d’haplotypes (rHAPs), au niveau du promoteur proximal, de 11 gènes codant pour des composantes de la voie intrinsèque de l’apoptose. Pour ce faire, nous avons sous-cloné les rHAPs majeurs dans un vecteur contenant le gène rapporteur luciférase (pGL3b). Ces constructions furent utilisées dans des essais de transfections transitoires dans 3 lignées cellulaires (Hela, Jeg3 et Jurkat). Nous avons observé qu’au moins 2 rHAPs influencent significativement l’activité transcriptionelle de façon allèle spécifique. Ces rHAPs sont associés aux gènes YWHAB et YWHAQ. Les analyses de retard sur gel d’électrophorèse (EMSA) ont permis d’identifier 2 sites de liaison ADN-protéine différentielles dans les rHAPs du gène YWHAB. La variabilité du niveau d’expression des gènes étudiés pourrait contribuer à la susceptibilité interindividuelle de développer un cancer, tel que la leucémie de l’enfant. / The regulation of apoptosis is important in maintaining cellular homeostasis and the integrity of the genetic material. Apoptosis is a cellular mechanism that eliminates damaged cells. The operation of this biological pathway is crucial to counter the spread of cells and their genetic abnormalities. Deregulation of genes encoding components of the intrinsic pathway of apoptosis is frequently observed in various types of cancers, including leukemia. We propose that functional polymorphism located in the regulatory region (rSNP) of genes involved in the intrinsic apoptosis pathway would have a significant impact in oncogenesis by altering the expression levels of these genes. In this study, we validated, using a combination of in silico and in vitro approaches, the functional impact of genetic variability (analyzed as haplotypes, rHap) at the proximal promoters of 11 genes encoding components of the intrinsic apoptosis pathway. To do this, we subcloned the major rHaps in a vector containing the luciferase reporter gene (pGL3b). These constructs were used in transient transfection assays in three human cell lines (HeLa, Jurkat and JEG3). We observed that at least 2 rHaps significantly influence the transcriptional activity of a specific allele. These rHaps are associated with genes YWHAB and YWHAQ. Electrophoretic mobility shift assays (EMSA) have identified 2 sites for differential DNA-protein binding with the rHaps of YWHAB. Variability in the level of expression of the genes studied could contribute to interindividual susceptibility to developing cancer, such as childhood leukemia.

La susceptibilité ou la résistance aux cancers peuvent impliquer plusieurs mécanismes, incluant l’apoptose, la croissance cellulaire et la différenciation, la réplication et la réparation de l’ADN. Mon projet porte plus particulièrement sur l’apoptose. Une dérégulation dans les voies d’activation de l’apoptose entraîne une accumulation de cellules déréglées, créant ainsi un environnement propice à l’instabilité génétique et au développement du cancer. Comme l’apoptose est une voie biologique hautement régulée, nous proposons l’hypothèse que des polymorphismes « fonctionnels » dans les régions de régulations des gènes (rSNPs) perturberaient cette voie à cause de taux variables de transcrits et des protéines correspondantes dû à la modification des sites de reconnaissances des facteurs de transcription. Les principaux objectifs de mon projet sont : (i) identifier les SNPs présents dans la région promotrice des gènes d’apoptose; (ii) déterminer les haplotypes de promoteurs les plus fréquents présents dans la population générale; (rHaps) (iii) vérifier leurs impacts fonctionnels sur l’expression génique par des essais in vitro (gène rapporteur et retard sur gel). Cette étude permettra d’identifier des rSNPs et rHaps ayant un impact sur le niveau d’expression des gènes d’apoptose, au moins dans un contexte in vitro. Ces différences alléliques au niveau de l’expression de ces gènes d’apoptose pourraient contribuer à la susceptibilité interindividuelle de développer un cancer. / Cancer susceptibility can involve many different mechanisms, including apoptosis, cell growth, cell differentiation, DNA replication and DNA repair. My project focuses on the apoptosis pathway. Decreased apoptosis level can lead to the accumulation of dysregulated cells, creating a favourable environment for genetic instability and tumorigenesis. Since apoptosis is a highly regulated pathway, the hypothesis of this project is that functional polymorphisms (rSNPs) in the regulatory regions of apoptosis genes such as promoters could create or disrupt transcription factor binding sites and modify their transcription levels. The main objectives of my project are: (i) Identify rSNPs in promoter regions of apoptosis genes; (ii) Calculate the frequent promoter haplotypes (rHaps) in general population; (iii) Validate their functional impact on gene expression with in vitro assays (gene reporter and gel shift). This study will allow identification of rSNPs and rHaps that could influence apoptosis gene expression levels, at least in the in vitro context. These allelic differences of expression in apoptosis genes could contribute to interindividual cancer susceptibility.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Durante os últimos anos a avicultura nacional tem sofrido constantes desafios, tendo como objetivo ampliar o mercado consumidor do frango de corte ao redor do mundo. Entretanto, cada mercado apresenta exigências diferentes, como no caso do mercado europeu que estabelece a exclusão dos antibióticos como promotores de crescimento na alimentação dos frangos de corte. Visando esta problemática, realizou-se este estudo, com o objetivo de analisar os resultados da utilização dos ácidos lático e butírico, isolados e associados, como aditivos em dietas de frangos de corte, em relação ao promotor de crescimento usualmente utilizado nas dietas dos mesmos. Foram avaliados: o desempenho, a imunidade humoral e a morfometria intestinal das aves. Utilizaram-se 1400 pintainhos machos da linhagem comercial Ross, divididos em cinco tratamentos (controle - sem aditivo -; ácido butírico; ácido lático; ácido butírico + ácido lático e avilamicina - antibiótico, promotor de crescimento tradicionalmente utilizado na produção de frangos de corte). Os resultados de desempenho indicaram que a interação dos ácidos foi significativa na fase inicial, entretanto não ocorreu um efeito aditivo dos ácidos, sendo o uso do ácido butírico isoladamente mais recomendável durante essa fase. Já na fase de crescimento, a interação foi significativa com um efeito aditivo, recomendando seu uso nas rações de crescimento. De acordo com os títulos médios de anticorpos obtidos no estudo, a interação foi significativa aos 35 dias de idade e mostrou um efeito sinérgico, sendo a combinação dos ácidos em questão um potente modulador da imunidade humoral. Os resultados obtidos nas análises de morfometria intestinal não foram conclusivos. Em termos gerais, requerem-se mais estudos quanto ao uso dos ácidos orgânicos como promotores de crescimento. / During the last years, the national poultry keeping has suffered constant challenges, having as objective to diversify the consuming market of the broiler to around of the world. However, each market presents different requirements, as in the case of the European market that establishes the exclusion of antibiotics as promotional of growth in the feeding of the broilers. Aiming at this problematic one, this study was become fullfilled, with the objective to analyze the results of the association of the butyric and lactic acids as additives in the ration of broilers, in comparison to the results gotten for the usually used promoters of growth in the diets of broilers. They had been evaluated: the animal performance, humoral immunology and intestinal morfometric of the birds. 1400 male chickens of the commercial ancestry Ross had been used, dividing them in five groups with different treatments to that if it relates to the additive use. Being a group it has controlled absent of additive, a group with butyric acid, a group with lactic acid, the fourth group with the association of butyric and lactic acids, and the fifth group with avilamicina (antibiotic) as promotional of traditionally used growth in the production of broilers. The performance results had indicated that the interaction of acid ones was significant in the initial phase, mean while did not occur an additive effect of acid ones, being the use of the butyric acid separately more recommendable during this phase. Already in the growth phase, the interaction was significant with an additive effect, recommending its use in the rations of growth. In accordance with the average headings of antibodies gotten in the study, the interaction was significant in the third sampling and showed a synergic effect of acid ones, being the combination of acids in question a powerful modulator of the humoral immunity. Meanwhile, the results gotten in the analyses of intestinal morfometric had not been conclusive. In general terms, one requires more organic acid studies that confirm the use of as the promotional ones of growth.