A eficiência da reprodução sexual de plantas depende do correto desenvolvimento dos órgãos sexuais: estame e pistilo. Mecanismos moleculares complexos controlam a proliferação e expansão celular que resultam no correto desenvolvimento destes órgãos. Em nosso laboratório foi identificado um gene preferencialmente expresso no pistilo de Nicotiana tabacum, o gene 092H06. Este gene codifica uma pequena proteína de 68 aminoácidos, e função desconhecida. Análises anteriores sugerem que o produto proteico do gene 092H06 seja responsável por inibir o processo de expansão celular nos órgãos reprodutivos (Brito,2010). Para compreender o papel deste gene, no desenvolvimento do pistilo, foram realizados experimentos de qRT-PCR para determinar se os níveis de expressão de genes para -expansina, -expansina, ciclina B1.2 e actina, ligados aos processos de divisão e expansão celular, em plantas transgênicas de silenciamento e superexpressão do gene 092H06. Foram realizadas análises morfológicas nos estigmas/estiletes e ovários das plantas transgênicas de segunda geração (T2), por microscopia óptica. Os resultados mostram uma tendência de aumento no volume das células tanto nas plantas transgênicas de silenciamento, como nas de superexpressão. Entretanto, nas plantas de silenciamento ocorreu um aumento visível das estruturas reprodutivas, o que não foi observado nas plantas de superexpressão. Adicionalmente, foram realizados experimentos de citometria de fluxo, para verificar a ocorrência de endorreduplicação. Os resultados mostraram que não ocorreu endorreduplicação nas células das plantas transgênicas. No screening de uma biblioteca de duplo híbrido, usando 092H06 como isca, foram encontrados 4 candidatos a parceiros de interação: 1) biotin/lipolyl attachmente domain-containing protein; 2) unknown protein; 3) trypsin proteinase inhibitor precursor e 4) RING/U-box. Para auxiliar no estudo da função do gene 092H06, a proteína recombinante 092H06-Histag foi produzida com sucesso, na forma solúvel, em E. coli. Os resultados alcançados neste trabalho contribuem para avançar o conhecimento sobre este novo gene expresso nos órgãos reprodutivos das plantas. / The efficiency of plant sexual reproduction depends on the correct development of the sexual organs: stamen and pistil. Complex molecular mechanisms control cell proliferation and expansion that result in the correct development of these organs. In our laboratory a gene preferentially expressed in Nicotiana tabacum pistil has identified, the 092H06 gene. This gene encodes a small protein of 68 amino acids of unknown function. Previous analyzes suggest that the protein product of the gene 092H06 is responsible for inhibiting the cell expansion process in the reproductive organs (Brito, 2010). To understand the role of this gene in pistil development, experiments of qRT-PCR to determinate the expression levels of the -expansins, -expansins, cyclin B1.2 and actin, genes which connected to the cell division and expansion processes, were carried out on transgenic plants silencing and overexpressing the 092H06 gene. Morphological analyzes on stigmas/styles and ovaries of second generation (T2) transgenic plants were performed by optical microscopy. The results show a tendency to increased cellular volume on the silencing transgenic plants, as well as on the overexpressing plants. However, in the silencing plants there was a visible increase of the reproductive structures, what has not been observed on the overexpressing plants. Additionally, flow cytometry experiments were carried out to verify the occurrence of endoreduplication. The results showed that no endoreduplication has occurred on the cells of the transgenic plants. The screening of a yeast two-hybrid assays, using 092H06 as bait, has found 4 interaction partners candidates: 1) biotin/lipolyl attachment domain-containing protein; 2) unknown protein; 3) trypsin proteinase inhibitor precursor and 4) RING/U-box. To assist the study of the 092H06 function, the recombinant 092H06-HIStag protein has been produced with success, in the soluble form, in E.col. The results obtained in this work contribute to advance the knowledge of this novel gene expressed on the plant reproductive organs.

Made available in DSpace on 2014-07-29T15:25:12Z (GMT). No. of bitstreams: 1 patricia kott.pdf: 7658177 bytes, checksum: ad9ad7831fb8596c7e55d3b1f0eee117 (MD5) Previous issue date: 2010-02-26 / Paracoccidioides brasiliensis is a thermo-dimorphic human pathogenic fungus that lives at 23”C in the mycelium phase (infecting phase) and at 37”C in the yeast phase (parasite phase). In attempt to survive, the cell wall of fungi can change its composition and/or structure in response to environmental stress by compensatory mechanisms. The molecules involved in these mechanisms are possible target for the development of effective antifungal agents. In P. brasiliensis, the main components of the cell wall are glucans and chitin polymers. These polymers make a primary barrier that is responsible for the structural integrity and form of the cell wall. In this work the behavior of P. brasiliensis was evaluated against stress conditions with the aim of study, for the first time, the mechanisms used by this fungus in the maintenance of the cell wall integrity. Our results shown that P. brasiliensis yeast cells are sensitive to cell wall stressors calcofluor white (CFW), congo red (CR), SDS, KCl, NaCl and sorbitol. There was an increase in the PbFKS1 transcripts expression and in the content of cell wall β- 1,3-glicana after treatment with all stressor agents. After treatment with SDS and KCl the PbGFA1 transcripts expression and the cell wall GlcNAc residues also increased. The transcript expression of PbGEL3 was also evaluated being increased after treatment with CFW, NaCl and sorbitol. Thus we showed that these molecules are involved in the maintenance of the cell wall against stress conditions. Apart from these analyses we obtained the active recombinant protein PbFks1pc. Through the anti-PbFks1pc antibody we performed immunocitolocalization assays. These experiments revealed the localization of PbFks1p in regions of apical growth in the mycelium phase and in the cell surface in yeast phase. / Paracoccidioides brasiliensis Š um fungo patog‰nico e termodim‚rfico que se apresenta na forma de micŠlio a 23”C (forma infectante) e na forma de levedura a 37”C (forma parasit€ria). A parede celular de fungos Š capaz de alterar sua composiƒ o e atŠ mesmo sua estrutura em resposta a condiƒ•es ambientais de estresse atravŠs de mecanismos compensat‚rios. As prote…nas envolvidas nestes mecanismos s o poss…veis alvos para o desenvolvimento de agentes antifˆngicos espec…ficos. Em P. brasiliensis, os principais pol…meros que constituem a parede celular s o glicanas e quitina. Estes pol…meros formam uma barreira prim€ria que Š respons€vel pela integridade estrutural e forma da parede celular. Neste trabalho, o comportamento de P. brasiliensis diante de situaƒ•es de estresse foi investigado com o intuito de estudar, pela primeira vez, os mecanismos utilizados por este fungo na manutenƒ o da integridade da parede celular. Nossos resultados mostram que cŠlulas leveduriformes de P. brasiliensis s o sens…veis aos agentes estressores da parede celular calcofluor white (CFW), congo red (CR), SDS, NaCl, KCl e sorbitol. Houve um aumento na express o de transcritos de PbFKS1 e no conteˆdo do pol…mero de β-1,3-glicana na parede celular ap‚s tratamento com todos os agentes estressores. Ap‚s tratamento com SDS e KCl a express o de transcritos de PbGFA1 assim como o conteˆdo de res…duos de GlcNAc na parede celular tambŠm aumentaram. A express o dos transcritos de PbGEL3 tambŠm foi avaliada estando aumentada ap‚s tratamento com CFW, NaCl e sorbitol. Desta forma, mostramos que estas molŠculas est o envolvidas na manutenƒ o da parede celular diante de situaƒ•es de estresse. AlŠm destas an€lises, a prote…na recombinante PbFks1pc ativa foi obtida. AtravŠs do anticorpo anti-PbFks1pc realizamos ensaios de imunocitolocalizaƒ o. Estes experimentos revelaram a presenƒa de PbFks1p em regi•es de crescimento apical no micŠlio e na superf…cie celular em cŠlulas leveduriformes.

Plant secondary products such as flavonoids have a variety of roles in plants including UV protection, antifeedant activity, pollinator attraction, stress response, flavor, and many more. These compounds also have effects on human physiology. Glucosylation is an important modification of many flavonoids and other plant secondary products. In grapefruit, glucosylation is important in the synthesis of the bitter compound naringin and several flavonoid glucosyltransferase (GT) enzymes have been characterized from young grapefruit leaf tissue. To study structure and function of flavonoid GTs, it is necessary to isolate cDNA’s that can be cloned and manipulated. In prior work, the plant secondary product glucosyltransferase (PSPG) box was used to identify putative GT clones. We report on results from experiments to test the hypothesis that PGT clones 4 and 11 are plant secondary product GTs, specifically flavonoid GTs. Previously, PGT 4 was cloned into a bacterial expression system, however all protein was localized into inclusion bodies and GT activity could not be tested. For this work, recombinant PGT 4 and PGT 11 were transformed into yeast and the proteins expressed and screened for glucosyltransferase activity with a variety of flavonoid substrates including flavanones, flavones, and flavonols.

S-layer proteins of S. ureae ATCC 13881 form on the cell surface an S-layer lattice with p4 square type symmetry and a period of about 13.5 nm. These lattices were shown to be the excellent nanotemplates for deposition of regular metal clusters. The synthesis of the S. ureae S-layer protein is highly efficient, the protein accounts for approximately 10-15 % of the total cell protein content, judged by the SDS-PAGE results. Besides, the S-layer protein production is tightly regulated, since only negligible amounts of S-layer proteins are observed in the medium at different cell growth phases. At the same time, mechanisms of the regulation of S-layer protein synthesis are poorly understood. As several hundreds of S-layer proteins are produced per second during the cell growth, the S-layer gene promoters are among the strongest prokaryotic promoters at all. However, little is known about factors regulating the expression of S-layer genes, furthermore, no experimental identification of other upstream regulatory sequences except for -35/-10 and RBS sequences was presented to our knowledge to date. A sequence of the S-layer gene of S. ureae ATCC 13881, encoding the previously described S-layer protein, was identified in this work by combination of different approaches. The largest part of the gene, excluding its upstream regulatory and ORF 5’ regions, was isolated from a genomic library by hybridization. The sequence of the isolated fragment proved to contain additionally an 1.9 kb non-coding region and an incomplete 0.8 kb ORF region in its 3’-part. No RBS sequence and apparent promoter regions could be identified in front of the latter sequence, suggesting that it might represent a pseudogene sequence. The sequences of the 5’ and upstream regions of the S. ureae ATCC 13881 S-layer gene were identified by combination of PCR-sequencing and chromosome walking. Totally, a sequence of the 6.4 kb long region of S. ureae genomic DNA was established. The sequence of the S. ureae S-layer protein was deduced from the respective gene sequence and agreed with the peptide sequences, obtained after N-terminal sequencing of tryptic peptides of the S. ureae ATCC 13881 S-layer protein. For the protein the name SslA was proposed, which is an abbreviation for “Sporosarcina ureae S-layer protein A”. Several specific features were observed in gene organisation of sslA, which are also characteristic for other S-layer genes. The distance between the -35/-10 region and the ATG initiation codon is unusually long and a 41 bp palindromic sequence is present in the immediate vicinity of the -35/-10 region. Besides, a distant location of the rho-independent transcription terminator, which is 647 bp remote from the stop codon, will result in the mRNA transcripts with unusually long trailer region. Both the long 5’ UTR and the long 3’ trailer may have a regulatory function, either by conferring increased mRNA stability and/or by affecting translation efficiency. Potentially these sequences may define the binding sites of regulatory proteins. For example, palindromic sequences constitute the regulatory sites in several bacterial operons and may act as the binding sites of regulatory dimeric proteins. In respect to the conservation of the sslA sequence high similarity to the sequences of other functional S-layer genes, especially the slfA and slfB genes of B. sphaericus, was observed, whereas the results of phylogenetic analysis support the hypothesis that S-layer genes may have evolved via the lateral gene transfer. Based on the sslA sequence, several recombinant proteins with truncations of the terminal protein parts or C-terminal fusion of either EGFP or histidine tags were constructed. For all the truncated or EGFP-fusion SslA derivatives high level overexpression in E. coli was possible. For native SslA a moderate level of expression was observed suggesting that its high intracellular concentration may downregulate the protein synthesis. Interestingly, fluorescence microscopy indicates the same intracellular localization for heterologously produced recombinant proteins with fusions of EGFP either to the precursor or to the native SslA protein, suggesting that SslA secretion signal is not functional in E. coli. Heterologously produced SslA derivatives with truncations of N-, C- or both N- and C-terminal parts were shown to self- assemble in vitro, although the size of self-assembly structures was different from that observed upon the self-assembly of the native SslA. In the latter case extended self-assembly layers with the size up to 5x10 µm were observed, with a surface area of up to two orders of magnitude higher than that of S-layer patches, routinely isolated from S. ureae surface. Dependent on the applied recrystallization conditions preferential formation of single- or multilayer self-assembly structures was observed.

Made available in DSpace on 2016-06-02T20:20:34Z (GMT). No. of bitstreams: 1 4606.pdf: 10556895 bytes, checksum: debf5633a053f88ca7dd4618edef9175 (MD5) Previous issue date: 2012-07-06 / Universidade Federal de Minas Gerais / The plant biomass is a large-scale available resource and one of its more important applications is the second-generation ethanol production. However, the enzyme cost is one of the biggest barriers for economically viable ethanol from biomass. Therefore, it is important to identify fungal strains that can produce high concentrations of plant biomass-degrading enzymes. The aim of this work was to clone, study the gene expression and characterize the plant biomass-degrading transcript set of the filamentous fungus Trichoderma harzianum IOC-3844. A total of 1,543 highquality reads from the Trichoderma harzianum IOC-3844 cellulose induced cDNA library were organized into 1,002 transcripts representing 167 contigs and 835 singlets. Of these 1,002 transcripts 646 had unknown functions and 356 showed associated functions. Among the transcripts with associated functions, we found 20 transcripts related to plant biomass deconstruction. The real time PCR analysis of Trichoderma harzianum IOC-3844 mycelia grown for 36 and 60 hours in cellulose, revealed that the levels of the following mRNAs were induced by at least 2,000-fold when compared to uninduced mycelia: cbh1, cbh2, egl1, egl2, egl3, egl7 and swo1. In some cases, the values were higher than 100,000-fold. Among the transcripts analyzed by real time PCR, cbh1, cbh2 and egl7 exhibited the highest expression levels. The Trichoderma harzianum IOC-3844 exhibited a repertoire with high expression of plant biomassdegrading transcripts. The enzymes EGIII and Xyn2 were recombinantly expressed in Pichia pastoris, showing good quality purification and good enzymatic activity. The heterologous expression assays made possible future studies aiming at the industrial application of the enzymes. Therefore, this strain showed potencial to produce biomassdegrading enzymes for second-generation ethanol production and to be a source of enzymes for the paper industry / A biomassa vegetal é um recurso disponível em larga escala e uma das suas mais imporantes aplicações é a produção de etanol de segunda geração. No entanto, o custo das enzimas é um dos maiores entraves para a produção economicamente viável deste etanol. Neste contexto, é importante encontrar organismos produtores de grandes quantidades de enzimas que degradam a biomassa. Os objetivos deste estudo foram clonar, estudar a expressão gênica e caracterizar o conjunto de enzimas que degradam a biomassa vegetal, do fungo filamentoso Trichoderma harzianum IOC-3844. Um total de 1.543 seqüências de boa qualidade, geradas a partir de uma biblioteca de cDNA do Trichoderma harzianum IOC-3844, induzido por celulose, foi organizado em 1.002 transcritos, sendo 167 representados por mais de uma seqüência e 835 representados por apenas uma seqüência. Destes transcritos, 356 tiveram função associada e 646 não tiveram. Com isso, entre os transcritos com função associada, foram listados 20 transcritos envolvidos com degradação de biomassa vegetal. Análises de PCR em tempo real do micélio de Trichoderma harzianum IOC-3844, crescido por 36 e 60 horas em celulose, mostraram níveis de mRNA mais de 2.000 vezes mais representados para os transcritos cbh1, cbh2, egl1, egl2, egl3, egl7 e swo1, quando comparados com o micélio não induzido. Em alguns casos as maiores representatividades alcançaram valores superiores a 100.000 vezes. Entre os transcritos analisados o cbh1, o cbh2 e o egl7, mostraram os mais altos níveis de expressão. O Trichoderma harzianum IOC-3844 exibiu um repertório com alta expressão de transcritos envolvidas na degradação de biomassa vegetal. As enzimas EGIII e Xyn2 foram expressas em sistema recombinante com uso da levedura Pichia pastoris, apresentando facilidade de purificação e boa atividade enzimática. Os ensaios de expressão heteróloga viabilizaram estudos posteriores que visam a aplicação industrial das enzimas. Assim, esta cepa mostrou potencial para produzir enzimas que degradam a biomassa para a produção de etanol de segunda geração, e para ser fonte de enzimas para a indústria de papel.

A eficiência da reprodução sexual de plantas depende do correto desenvolvimento dos órgãos sexuais: estame e pistilo. Mecanismos moleculares complexos controlam a proliferação e expansão celular que resultam no correto desenvolvimento destes órgãos. Em nosso laboratório foi identificado um gene preferencialmente expresso no pistilo de Nicotiana tabacum, o gene 092H06. Este gene codifica uma pequena proteína de 68 aminoácidos, e função desconhecida. Análises anteriores sugerem que o produto proteico do gene 092H06 seja responsável por inibir o processo de expansão celular nos órgãos reprodutivos (Brito,2010). Para compreender o papel deste gene, no desenvolvimento do pistilo, foram realizados experimentos de qRT-PCR para determinar se os níveis de expressão de genes para -expansina, -expansina, ciclina B1.2 e actina, ligados aos processos de divisão e expansão celular, em plantas transgênicas de silenciamento e superexpressão do gene 092H06. Foram realizadas análises morfológicas nos estigmas/estiletes e ovários das plantas transgênicas de segunda geração (T2), por microscopia óptica. Os resultados mostram uma tendência de aumento no volume das células tanto nas plantas transgênicas de silenciamento, como nas de superexpressão. Entretanto, nas plantas de silenciamento ocorreu um aumento visível das estruturas reprodutivas, o que não foi observado nas plantas de superexpressão. Adicionalmente, foram realizados experimentos de citometria de fluxo, para verificar a ocorrência de endorreduplicação. Os resultados mostraram que não ocorreu endorreduplicação nas células das plantas transgênicas. No screening de uma biblioteca de duplo híbrido, usando 092H06 como isca, foram encontrados 4 candidatos a parceiros de interação: 1) biotin/lipolyl attachmente domain-containing protein; 2) unknown protein; 3) trypsin proteinase inhibitor precursor e 4) RING/U-box. Para auxiliar no estudo da função do gene 092H06, a proteína recombinante 092H06-Histag foi produzida com sucesso, na forma solúvel, em E. coli. Os resultados alcançados neste trabalho contribuem para avançar o conhecimento sobre este novo gene expresso nos órgãos reprodutivos das plantas. / The efficiency of plant sexual reproduction depends on the correct development of the sexual organs: stamen and pistil. Complex molecular mechanisms control cell proliferation and expansion that result in the correct development of these organs. In our laboratory a gene preferentially expressed in Nicotiana tabacum pistil has identified, the 092H06 gene. This gene encodes a small protein of 68 amino acids of unknown function. Previous analyzes suggest that the protein product of the gene 092H06 is responsible for inhibiting the cell expansion process in the reproductive organs (Brito, 2010). To understand the role of this gene in pistil development, experiments of qRT-PCR to determinate the expression levels of the -expansins, -expansins, cyclin B1.2 and actin, genes which connected to the cell division and expansion processes, were carried out on transgenic plants silencing and overexpressing the 092H06 gene. Morphological analyzes on stigmas/styles and ovaries of second generation (T2) transgenic plants were performed by optical microscopy. The results show a tendency to increased cellular volume on the silencing transgenic plants, as well as on the overexpressing plants. However, in the silencing plants there was a visible increase of the reproductive structures, what has not been observed on the overexpressing plants. Additionally, flow cytometry experiments were carried out to verify the occurrence of endoreduplication. The results showed that no endoreduplication has occurred on the cells of the transgenic plants. The screening of a yeast two-hybrid assays, using 092H06 as bait, has found 4 interaction partners candidates: 1) biotin/lipolyl attachment domain-containing protein; 2) unknown protein; 3) trypsin proteinase inhibitor precursor and 4) RING/U-box. To assist the study of the 092H06 function, the recombinant 092H06-HIStag protein has been produced with success, in the soluble form, in E.col. The results obtained in this work contribute to advance the knowledge of this novel gene expressed on the plant reproductive organs.

Les anticoagulants antivitamine K (AVK) sont destinées à limiter la coagulation du sang. Ils sont donc susceptibles de provoquer des saignements. Les AVK ralentissent le cycle de la vitamine K qui est indispensable à la gamma-carboxylation de certaines protéines (PVKD). Les AVK inhibent l'activité vitamine K époxide reductase (VKOR), principalement catalysée par VKORC1. Ce sont des médicaments anticoagulants utilisés chez l'homme. Chez les rongeurs, ils servent de rodonticides. Une résistance aux AVK est observé tant chez l'homme que chez le rongeur.Chez des patients résistants aux AVK, 26 mutations ont été décrites dans la zone codante de VKORC1. L'expression hétérologue de ces enzymes mutées n'a permis de trouver que 6 mutations impliquées dans la résistance. Repérer ces mutations avant le début d'un traitement permettra une mise en place du traitement plus rapide. Les autres mutations ne seraient pas responsables du phénotype observé.La VKORC1L1 a été décrite comme une protéine agissant contre le stress oxydatif. Notre travail confirme que l'enzyme catalyse la réaction VKOR. Si sa participation dans la réduction de la vitamine K époxide est insignifiante dans le foie, il en est tout autrement dans les autres tissus testés. De plus, la VKORC1L1 apparait plus résistante aux AVK par rapport à la VKORC1. Ces propriétés catalytiques de la VKORC1L1 permettent d'expliquer l'absence d'effets des AVK sur les PVKD d'origine extra-hépatiques.Enfin, un travail de mutagénèse dirigée a permis d'abaisser ou d'augmenter considérablement la sensibilité de VKORC1L1 aux AVK. Ces résultats nous permettent de décrire l'implication de différents acides aminés dans l'interaction avec les AVK / Anticoagulant vitamin K antagonists (VKA) are molecules designed to prevent or delay blood clotting. They cause bleeding by slowing the recycling of vitamin K, an essential micronutrient for posttranslational modification of specific proteins (VKDP). It has been shown that VKA specifically inhibit VKORC1 enzyme which catalyze the VKOR reaction. VKA are used as rodenticides to control the proliferation of populations of pest rodents. In humans, they are used in the treatment and prevention of the occurrence of thromboembolic events. Due to the widespread use of these VKA, it was observed a phenomenon of resistance which is essential to better understand for economic, ecological or public health interests. In humans, 25 of 26 mutations were characterized. While these changes have been observed in patients resistant to VKA, the causality of these mutations has been demonstrated for 6 mutations. The ability to detect these changes before the start of treatment will allow the future implementation of the much faster and less expensive. Other mutations are not responsible for the observed phenotype.Moreover, VKORC1L1 has been described as an enzyme whose function is to act against oxidative stress. This study confirms that the enzyme catalyzes the VKOR reaction. If it appears that the liver in its participation in the reduction of vitamin K epoxide is insignificant, it is quite different in other tissues tested. In addition, VKORC1L1 appears more resistant to VKA over the VKORC1. Finally, directed mutagenesis of these residues lead to the decrease or the increase of VKORC1L1 sensitivity to VKA. These data result to the implication of residues in their interaction with VKA

Abstract In plants, there are at least three types of haemoglobins (Hbs): symbiotic, non-symbiotic and truncated. Symbiotic Hbs are known to participate in nitrogen fixation, but the roles of the latter two groups are more obscure. Previous reports have connected both plant non-symbiotic and truncated Hbs to the scavenging of an important signal molecule, nitric oxide (NO). The aim of the present thesis was to study the effects of a bacterial Hb of Vitreoscilla sp. (VHb) on a woody model organism, hybrid aspen (Populus tremula x tremuloides), and the role of endogenous hybrid aspen Hbs. To store the produced hybrid aspen lines, the suitability of different cryopreservation methods was also tested. VHb-expressing hybrid aspens were generated by Agrobacterium-mediated gene transfer. The effects of VHb expression were examined in standard greenhouse conditions, under elevated UV-B light as well as during culture with ectomycorrhizal fungi. Both slow cooling and vitrification methods were applied in cryostoring samples of the different genetic backgrounds. Hybrid aspen non-symbiotic and truncated Hb genes PttHb1 and PttTrHb, respectively, were also isolated. The function of the genes and corresponding proteins PttHb1 and PttTrHb were studied using non-transgenic and VHb hybrid aspen lines as well as a mutant yeast (Saccharomyces cerevisiae) defective in NO resistance. VHb expression did not improve the general growth of hybrid aspen but resulted in enhanced starch accumulation in chloroplasts, pointing to changes in energy metabolism. Of the studied cryopreservation protocols, the slow cooling of dormant in vivo buds proved to be the most feasible way of cryostoring hybrid aspen lines. The culture with the ectomycorrhizal fungus was shown to increase the expression of both PttHb1 and PttTrHb in the roots of non-transgenic lines. However, the fungi did not up-regulate the hybrid aspen Hb genes in the VHb lines. Therefore, it is hypothesized that endogenous Hbs may contribute to the growth of roots and that VHb may compensate this function. When expressed alone in the mutant yeast, the recombinant PttHb1 and PttTrHb did not protect cells against the toxicity of NO. Subsequently, a novel mRNA transcript of the heterotrophic ferredoxin NADP+ oxidoreductase gene PtthFNR was found. The absence of a plastid presequence in the transcript suggests targeting of the encoded protein into cytosol. The coexpression of PttHb1 and cytosolic PtthFNR partially rescued the mutant yeast during NO treatment, demonstrating for the first time that plant Hb1 with an applicable reductase scavenges NO in vivo at a physiologically relevant rate. This thesis extends current knowledge about plant Hbs and the effects of VHb on a phenotype of a tree. It also provides new information about plant ferredoxin reductase genes. / Tiivistelmä Kasvihemoglobiinit jaetaan symbioottisiin, ei-symbioottisiin sekä ns. katkaistuihin eli truncated-hemoglobiineihin. Symbioottisten hemoglobiinien tiedetään osallistuvan typen sitomiseen, kun taas kahden muun ryhmän toiminta tunnetaan heikosti. Aiemmissa tutkimuksissa ei-symbioottiset ja truncated-hemoglobiinit on yhdistetty tärkeän signaalimolekyylin, typpimonoksidin (NO), haitallisuuden vähentämiseen. Tämän työn tarkoituksena oli tutkia Vitreoscilla sp. -bakteerin VHb-hemoglobiinin vaikutuksia puuvartiseen mallikasviin, hybridihaapaan (Populus tremula x tremuloides), sekä hybridihaavan omien hemoglobiinien merkitystä. Lisäksi työssä kokeiltiin eri nestetyppisäilytysmenetelmiä hybridihaapalinjojen varastoimiseksi. VHb:tä ilmentävät hybridihaavat tuotettiin Agrobacterium-välitteisellä geeninsiirrolla. VHb:n vaikutuksia tutkittiin kasvihuoneolosuhteissa, kohotetussa UV-B-säteilyssä sekä sienijuurikasvatuksen aikana. Eri linjojen nestetyppisäilytykseen sovellettiin sekä hidasta että nopeaa jäähdytystä. Tutkimuksissa myös eristettiin hybridihaavan PttHb1- ja PttTrHb-hemoglobiinigeenit. Näiden geenien sekä niiden koodaamien PttHb1- ja PttTrHb-proteiinien toimintaa tutkittiin siirtogeenittömien ja VHb-hybridihaapalinjojen sekä NO-herkän hiivamutantin (Saccharomyces cerevisiae) avulla. VHb-ilmennys ei parantanut hybridihaavan kasvua, mutta lisäsi tärkkelyksen kertymistä viherhiukkasiin, mikä viittaa muutoksiin energia-aineenvaihdunnassa. Tutkituista menetelmistä parhaiten hybridihaapalinjojen nestetyppisäilytykseen soveltui lepovaiheessa olevien in vivo -silmujen hidas jäähdytys. Toisin kuin VHb-kasveissa, sienijuurikasvatus voimisti sekä PttHb1:n että PttTrHb:n ilmenemistä siirtogeenittömien hybridihaapojen juurissa. Tästä johtuen esitettiin, että hybridihaavan hemoglobiinit voivat vaikuttaa juurien kasvuun ja VHb pystyy korvaamaan tämän toiminnon. Kun PttHb1 ja PttTrHb ilmennettiin yksin mutanttihiivassa, proteiinit eivät suojanneet soluja NO:n myrkyllisyydeltä. Tämän jälkeen työssä kuvattiin heterotrofisen ferredoksiini-NADP+-oksidoreduktaasi-geenin PtthFNR:n uusi mRNA-muoto. Plastidiin ohjaavan sekvenssin puuttuminen geenituotteesta viittaa siihen, että mRNA:n koodaama proteiini sijoittuu solulimaan. PttHb1:n ja PtthFNR:n yhtäaikainen ilmentäminen mutanttihiivassa paransi solujen elossa säilymistä NO-käsittelyn aikana, mikä osoittaa ensimmäistä kertaa, että kasvin Hb1 pystyy sopivan reduktaasin kanssa vähentämään NO-pitoisuutta elävässä eliössä. Tämä työ laajentaa tietämystä kasvihemoglobiineista sekä VHb:n vaikutuksesta puiden ilmiasuun. Työ myös lisää tietoa kasvien ferredoksiinireduktaasi-geeneistä.

La bactérie lactique Oenococcus oeni qui fait partie de la flore d’intérêt du vin, est responsable de la fermentation malolactique. Au cours de son développement dans le vin, Oenococcus oeni est confronté à des conditions physicochimiques drastiques (présence d’éthanol, pH 3,5, basse température, présence de composés soufrés, …). Sa capacité à s’adapter à ces conditions défavorables en fait un bon modèle d’étude de la réponse à de multiples stress chez les bactéries lactiques (Guzzo et al., 2000). L’un des mécanismes de résistance d’O. oeni fait intervenir une protéine de choc thermique de faible masse moléculaire ou sHsp (small Heat shock protein) nommée Lo18. La protéine Lo18 possède une activité de chaperon ATP-indépendante. C'est-à-dire que son association avec une protéine en cours de dénaturation permet de protéger la protéine et d’empêcher son agrégation. De plus elle est capable de s’associer avec les bicouches lipidiques et de stabiliser la structure lipidique.Les sHsp se caractérisent par la présence d’une région d’environ 90 acides aminés appelée α-cristallin impliquée dans l’activité de chaperon moléculaire in vitro. En général, les extrémités N- et C- terminales jouent un rôle essentiel dans le processus d’oligomérisation qui est nécessaire à l’activité chaperon. Dans l’optique d’étudier la relation entre la structure et la fonction de la sHsp Lo18, son activité et son oligomérisation ont été caractérisées à différents pH. Les résultats ont montré que le pH influe sur l’oligomérisation de Lo18 et également son activité de chaperon moléculaire. Des protéines Lo18 modifiées dans le domaine α-cristallin ont également été caractérisées. Elles ont permis de démontrer qu’une substitution d’acide aminé dans ce domaine altère l’activité de Lo18. Enfin des formes tronquées de Lo18 pour ses deux portions N- et C- terminales ont été construites, surproduites chez Escherichia coli, puis purifiées par chromatographie d’affinité hydrophobe.La capacité de Lo18 à empêcher l’agrégation des protéines et à stabiliser les membranes lipidiques nous a conduit à tester l’impact de Lo18 d’une part sur la surproduction in vivo chez Escherichia coli de protéines hétérologues d’intérêt, et d’autre part sur la formation d’un caillé laitier riche en caséine et lipides.La surproduction hétérologue de protéines chez E. coli est utilisée pour produire de grandes quantités de protéines à faibles couts. Cependant cette production n’est pas toujours efficace car l’accumulation d’une même protéine dans la cellule de la bactérie conduit souvent à son agrégation et à sa dégradation. Il apparait nécessaire de développer des systèmes permettant d’améliorer la solubilité des protéines surproduites chez E. coli. Nous avons donc testé les potentialités de Lo18 dans ce système, et montré une augmentation de la solubilité de protéines d’intérêt coproduites avec la sHsp Lo18 et/ou la Hsp GroEL/ES.Le lait comporte quatre composants dominants : l’eau, les matières grasses, les protéines et le lactose. En technologie fromagère, la coagulation correspond à une déstabilisation de l’état micellaire des protéines majoritaires du lait: les caséines. La prise en gel est suivie d’une phase d'égouttage, la synérèse, qui correspond à la perte d’une partie du lactosérum hors du gel. Les propriétés de chaperon moléculaire de la protéine Lo18 ont permis d’influencer l’agrégation des caséines in vitro. Nous avons donc appliqué Lo18 au modèle caillé laitier et décelé des applications industrielles possibles. Nous avons notamment montré en laboratoire une accélération de la phase de prise en gel, et une accélération du processus de synérèse. En modèle fromager nous avons mis en évidence que Lo18 permet de diminuer le taux d’humidité dans les fromages de type « pâtes molles » / The lactic acid bacteria Oenococcus oeni is part of the flora of interest in wine. It is responsible for malolactic fermentation. During its development in the wine, Oenococcus oeni is facing drastic physicochemical conditions (presence of ethanol, pH 3.5, low temperature, presence of sulfuric compounds). Its ability to adapt to these conditions makes of it a good model to study the response to multiple stress in lactic acid bacteria (Guzzo et al., 2000). One mechanism of resistance of O. oeni involves a Heat shock protein (Hsp) of low molecular weight or sHsp (small Heat shock protein) called Lo18.Lo18 protein has a chaperone activity ATP-independent. It is to say that its association with a protein during denaturation can protect the protein and prevent its aggregation. In addition Lo18 is able to bind with lipid bilayers and stabilize the lipidic structure.The sHsp are characterized by the presence of a region of about 90 amino acids, called α-crystallin, involved in molecular chaperone activity in vitro. In most cases, the N-and C-termini regions play an essential role in the oligomerization process that is necessary for the chaperone activity.In order to study the relationship between structure and function of Lo18, its activity and oligomerization were characterized at different pH. The results showed that the pH affects the oligomerization of Lo18 and also its molecular chaperone activity. Lo18 modified proteins in their α-crystallin region was also characterized. They have shown that a single amino acid substitution alters the activity of Lo18. Finally truncated forms of Lo18 in its two portions N-and C-termini were constructed, overproduced in Escherichia coli and purified by hydrophobic affinity chromatography.The ability of Lo18 to prevent aggregation of proteins and stabilize lipid membranes led us to test the impact of Lo18 for heterologous overproduction in Escherichia coli, and also in the formation of a curd milk rich in casein and fat.Overproduction of heterologous proteins in E. coli is widely used to produce large amounts of protein at low cost. However, this production is not easy because the accumulation of a protein in bacteria’s cell often leads to its aggregation and degradation. It appears necessary to develop systems to improve the solubility of proteins overproduced in E. coli. We therefore tested the potential of Lo18 in this system, and showed an increase in the solubility of proteins of interest coproduced with the sHsp Lo18 and / or the Hsp system GroEL / ES.Milk has four dominant components: water, fat, protein and lactose. In cheese technology, coagulation is a destabilization of the micellar state of the major proteins of milk: the caseins. Jellyfication phase is followed by a dripping phase called syneresis, which corresponds to the loss of part of the whey out of the gel.The properties of the sHsp Lo18 influenced the aggregation of the caseins in vitro. So we applied Lo18 on the curd milk model and detected possible industrial applications. In particular, we showed in laboratory an acceleration of the jellyfication phase, and an acceleration of the syneresis. In cheese model we have shown that Lo18 is able to reduce the humidity rate in cheeses

A hepatite viral causada pelo vírus da hepatite C (HCV) é um problema à saúde mundial e afeta cerca de 170 milhões de pessoas. O caso crônico da doença resulta em cirrose hepática e a maioria dos pacientes tratados não desenvolve uma resposta imune satisfatória. A proteína não estrutural 3 (NS3) pode estimular uma resposta celular que auxilia a resposta nos infectados. Nosso trabalho desenvolveu duas pseudopartículas virais que carregam um material genético para a protease da NS3 do HCV. Um dos sistemas, a HCVpp é constituída por proteínas do vírus da leucemia murina e outras do HCV. E o outro sistema, a partícula viral é baseada no Semliki Forest Virus (SFV). As células HEK293T e BHK-21 foram transfectadas para a formação das pseudopartículas HCVpp-NS3p1a e SFV-NS3p1a, respectivamente. Essas partículas foram quantificadas pela presença do material genético da NS3 por qRT-PCR, atingindo uma produção aproximada de 4x105 partículas HCVpp-NS3p1a/mL e 2,5x107 partículas SFV-NS3p1a/mL. Essas partículas foram utilizadas para infecção de células HuH-7.0 e BHK-21. / Viral hepatitis caused by the hepatitis C virus (HCV) is a global health problem, affecting about 170 million people. The chronic case of the disease results in liver cirrhosis and most patients do not develop a satisfactory immune response. The nonstructural protein 3 (NS3) can stimulate a cellular response that helps answer the infected. Our work has developed two viral pseudoparticles who carry a genetic material for the HCV NS3 protease. One of the systems is constituted by the HCVpp proteins of murine leukemia virus and other HCV. The other system, the viral particle is based on the Semliki Forest Virus (SFV). The HEK293T and BHK-21 cells were transfected for forming the pseudoparticles HCVpp-NS3p1a and SFV-NS3p1a, respectively. These particles were quantified by the presence of genetic material of NS3 by qRT-PCR, reaching a production of about 4x105 HCVpp-NS3p1a particles/mL and 2,5 x107 SFV-NS3p1a particles /mL. These particles were used for infection of Huh-7.0 cells and BHK-21.