O principal mecanismo de defesa das plantas frente a uma infecção viral é baseado no fenômeno chamado interferência por RNA (RNAi). Por meio da ação coordenada de proteínas como Argonautas, Dicers, RNA polimerases dependentes de RNA e proteínas de ligação a RNA de dupla fita (DRBs), o RNA viral é reconhecido e clivado a pequenos RNAs de interferência derivados de vírus (vsiRNAs). Os vsiRNAs, acoplados ao complexo proteíco de indução ao silenciamento, atuam sobre sequências de RNA ou DNA virais, podendo promover a clivagem, inibição da tradução ou metilação de seus alvos (para vírus de DNA). Neste trabalho foram realizados dois estudos que abordam mecanismos de defesa baseados em RNAi em plantas. No primeiro capítulo é descrito a integração do RNA1 do Cumcumber mosaic virus (CMV) no genoma de Glycine max. Através da análise de bibliotecas de sequenciamento de alta eficiência de RNA mensageiros (mRNAs), pequenos RNAs e DNAs de diferentes cultivares e diferentes tecidos de soja foi possível identificar que o evento de integração envolveu duas moléculas do RNA1 do CMV, o RNA de um retrotransposon e um mRNA de um gene endógeno. No locus aonde ocorreu esta integração as duas sequências do RNA1 estão em sentidos opostos. Os pequenos RNAs (sRNAs) das nossas bibliotecas alinham majoritariamente na região do RNA1 do CMV e são praticamente ausentes nas outras regiões da sequência integrada, sugerindo fortemente a formação de um grampo aonde hibridizam ambas as sequências do CMV. A presença desses sRNAs derivados do CMV em todos os tecidos estudados sugere uma provável função antiviral dessa sequencia que foi integrada em soja. No segundo capítulo, por microscopia confocal, foi estudada a interação entre as proteínas DRBs e o Potato virus X (PVX) durante a infecção viral em Nicotiana benthamiana. É demonstrado que as DRBs 2, 3 e 5 se realocam de sua posição original e se concentram em estruturas chamadas complexos de replicação viral durante a infecção por PVX. Esse fenômeno é um indicativo que essas proteínas podem estar atuando nos primeiros estágios de defesa da planta frente ao vírus. / The main defense mechanism of plants facing a viral infection is based on the phenomenon called RNA interference (RNAi). Through the coordinated action of proteins such as Argonaut, Dicers, RNA dependent RNA polymerases and double-stranded RNAbinding proteins (DRBs), the viral RNA is recognized and cleaved to virus-derived interfering small RNAs (vsiRNAs). The vsiRNAs, coupled to a protein complex that induce the silencing, act on DNA or RNA viral sequences promoting cleavage, translational inhibition or methylation of their targets (for DNA viruses). This work described two studies that address new defense mechanisms based on RNAi in plants. In the first chapter of this thesis is described the integration of the cucumber mosaic virus (CMV) RNA1 in the genome of Glycine max. Through the analysis of deep sequencing libraries of messenger RNA, small RNAs and DNA from different cultivars and different soybean tissues it was possible to identified that the integration event involved two molecules of CMV RNA1, the RNA of a retrotransposon and the mRNA of an endogenous gene. In the locus where the integration occurred the two RNA1 sequences are in opposite directions. Small RNAs (sRNAs) from our libraries mostly aligned in the region of CMV RNA1 and are practically absent in other regions of the integrated sequence, strongly suggesting the formation of a hairpin where both CMV sequences hybridize. The presence of these CMV-derived sRNAs in all surveyed tissues suggests a probable antiviral function for the sequence that was integrated into soybeans. In the second chapter, the interaction between the DRB proteins and the Potato virus X (PVX) during viral infection in Nicotiana benthamiana is assessed by confocal microscopy. It is shown that the DRBs 2, 3 and 5 relocate from its original position and concentrated in structures called viral replication complexes during infection by PVX. This is an indication that these proteins can act in the early stages of plant defense against the virus.

Braunschweig, Techn. Universiẗat, Diss., 2000.

Devant des besoins grandissants en produits biopharmaceutiques pour la santé humaine, les plateformes d'expression végétales proposent une alternative prometteuse aux approches classiques pour la production efficiente de protéines recombinantes. Alors que des études récentes mettaient en lumière l'importance des conditions de culture sur le rendement en protéines dans les plantes, l'impact d'un mode de culture hydroponique sur l'accumulation de ces protéines demeure méconnu, notamment pour l'hôte d'expression Nicotiana benthamiana utilisé couramment pour l'expression de protéines d'intérêt médical. Jusqu'ici, les études s'intéressant à l'impact des conditions de culture ont considéré des plants cultivés en pots alors que l'industrie s'oriente désormais vers des systèmes culturaux hydroponiques mieux adaptés à l'automatisation des procédures et à une utilisation plus rationnelle des ressources du milieu. Dans ce contexte, nous avons testé ici l'hypothèse de recherche selon laquelle un mode de culture hydroponique influence la distribution et l'accumulation d'un vaccin antigrippal, l'hémagglutinine H1, dans N. benthamiana. L'objectif général du projet était de cartographier le patron d'accumulation de l'antigène H1 dans la plante cultivée sous différentes conditions, incluant le mode de culture à proprement parler, différentes densités de culture et des intensités lumineuses variables. En bref, nos résultats indiquent qu'une plante cultivée en gouttières hydroponiques montre une biomasse foliaire redistribuée vers le sommet et un rendement en H1 supérieur comparé aux plantes cultivées en pots. Une densité de culture élevée en mode hydroponique a peu d'incidence sur la production de biomasse foliaire et le rendement en H1 à l'échelle de la plante, y compris lorsqu'exprimé par unité de surface de serre. Enfin, une intensité lumineuse élevée pendant la phase de croissance des plantes en cultures hydroponiques conduit à une biomasse foliaire plus importante avant l'étape d'expression protéique à proprement parler, sans influence marquée toutefois sur le rendement spécifique en antigène H1 dans les feuilles.

Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-06-14T10:48:13Z No. of bitstreams: 1 marinnagomes.pdf: 1050831 bytes, checksum: 19f0caa7f0448c9ddc9f79c839e5aa28 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-06-14T11:32:30Z (GMT) No. of bitstreams: 1 marinnagomes.pdf: 1050831 bytes, checksum: 19f0caa7f0448c9ddc9f79c839e5aa28 (MD5) / Made available in DSpace on 2018-06-14T11:32:30Z (GMT). No. of bitstreams: 1 marinnagomes.pdf: 1050831 bytes, checksum: 19f0caa7f0448c9ddc9f79c839e5aa28 (MD5) Previous issue date: 2018-02-21 / Acidentes ofídicos representam um grave problema de saúde pública devido a sua alta incidência e a gravidade dos seus efeitos. No Brasil, o gênero Bothrops é responsável por 85% dos casos de acidentes. A principal forma de tratamento para tais acidentes é feita através da soroterapia, utilizando-se anticorpos eqüinos, porém os mesmos não são capazes de neutralizar os efeitos sistêmicos do envenenamento e podem causar reações adversas. Uma nova perspectiva para o tratamento de acidentes ofídicos é a utilização de anticorpos recombinantes, mais precisamente a porção scFv. O fragmento scFvBap1 é oriundo de um anticorpo monoclonal, produzido em Escherichia coli foi capaz de neutralizar os efeitos hemorrágicos do envenenamento. Atualmente as plantas vêm sendo amplamente utilizadas na produção de proteínas recombinantes de interesse farmacêutico. Neste estudo, objetivou-se a expressão do fragmento scFv em Nicotiana benthamiana, a fim de avaliar seu potencial para ser utilizado como soro antiofídico. Foram avaliadas a expressão transiente do fragmento scFvBap1 em folhas de N. benthamiana, bem como a expressão estável na mesma planta. Os fragmentos produzidos foram capazes de reconhecer e neutralizar as toxinas BaP1 de B. asper, BnP1 de B. newidae e ATX de B. atrox, demonstrando seu potencial para ser utilizado na terapia contra envenenamentos ofídicos. O sistema de produção estável apresentou-se maior rendimento de proteínas (270 μg/g) quando comparado ao sistema de produção transiente (43 μg/g). Por fim, o sistema mostrou-se uma ótima alternativa para produção do fragmento de anticorpo quando comparado ao sistema bacteriano. / Ophidian accidents are a global health problem due to its high incidence and gravity of its effects. In Brazil, the genus Bothrops is responsible for 85% of the cases of accidents. The main form of treatment for snake envenoming is serum therapy using equine antibodies, however, they are not able to neutralize the local effects of the toxin and may also cause adverse reactions. A new perspective for the treatment of snakebite envenoming has emerged with the use of recombinant antibodies, particularly the Fv fragment (scFv). The scFvBap1, is an efficient antibody capable of neutralizing the hemorrhagic effects and proteolytic activity caused by metalloproteinases. Currently the plants have been widely used in the production of recombinant proteins of pharmaceutical interest. In this study, we aimed the expression of the scFv fragment in Nicotiana benthamiana, in order to evaluate its potential as an antiophiidic serum. The transient expression of the scFvBap1 fragment in leaves of N. benthamiana, as well as the stable expression in the same plant, were evaluated.The fragments produced were able to recognize and neutralize the toxins BaP1 from B. asper, BnP1 from B. newidae and ATX from B. atrox, demonstrating their potential to be used in therapy against ophidian poisoning. The stable production system presented higher protein yield (270 μg / g) when compared to the transient production system (43 μg / g). Finally, the system proved to be a good alternative for producing the antibody fragment when compared to the bacterial system.

Tableau d'honneur de la Faculté des études supérieures et postdorales, 2015-2016 / La moléculture végétale est une approche prometteuse pour la production de protéines d’intérêt médical ou industriel. Considérant les variations de rendement possibles dans une plante soumise à différentes conditions culturales, nos objectifs étaient : (i) de cartographier l’accumulation d’un antigène viral d’intérêt clinique dans les feuilles du tabac sauvage Nicotiana benthamiana utilisé comme bio-usine, et (ii) d’évaluer l’impact de la lumière en période de croissance sur le rendement total en antigène. Nous avons étudié les relations entre l’âge foliaire, le régime lumineux, l’expression du transgène et le rendement final en antigène dans les feuilles. Nos données confirment l’influence de l’âge sur les variations de rendement d’une feuille à l’autre, et l’impact positif de l’intensité lumineuse sur le rendement par plante. Elles mettent aussi en relief l’importance des tiges secondaires sur le rendement et le rôle clé de la transcription du transgène sur la teneur en antigène à l’échelle cellulaire. / Plant molecular farming is a promising approach to produce proteins of medical or industrial interest. Considering possible variations of protein yield in plants exposed to different cultural conditions, our goals were : (i) to map the accumulation of a clinically useful viral antigen in leaves of the tobacco relative Nicotiana benthamiana used as protein biofactory, and (ii) to evaluate the impact of light conditions during plant growth on total antigen yield. We looked at eventual relations in planta between leaf age, light intensity, light quality, transgene expression and final protein yield in leaves. Our data confirmed the central influence of leaf age on antigen yield variation from one leaf to another, and the positive impact of light intensity on total yield per plant. They also highlight the importance of secondary stem growth on total yield, and the key role of transgene transcription on antigen content at the cell scale.

Au niveau de la santé publique, l'impact meurtrier des infections au rotavirus humain (RVH) et les limites d'application des vaccins disponibles déterminent l'urgence et la nécessité de développer de nouvelles approches pour immuniser une grande partie de la population mondiale sans effets secondaires néfastes. Pour pallier à l'énorme demande en médicaments et en vaccins, plusieurs groupes de recherche tentent de développer de nouvelles méthodes de production à base de plantes transgéniques. La production de médicaments et vaccins chez les végétaux possèdent des avantages inhérents dont une absence de pathogènes humain, une inoculation facile, de faibles coûts de production à grande échelle et la production d'inocula bruts stockables. Le succès des systèmes d'expression à base de plantes justifie le développement de plants qui produisent beaucoup de biomasse et se transforment génétiquement de façon courante. Le travail présenté ici démontre le potentiel du système d'expression transitoire par agroinfiltration de Nicotiana benthamiana pour produire les antigènes VP7 et VP4∆ du RVH ainsi que l'adjuvant protéinique fljB de Salmonella typhimurium. Le clonage des séquences codantes des antigènes VP7, VP4∆ et fljB a permis de générer de multiples constructions simple, double et triple. Toutes les contructions ont été exprimées dans les feuilles agroinfiltrées à l'exception de VP4∆::VP7 qui n'a pas été détectée par immunobuvardage. Le rendement de protéines d'intérêt dans les extraits végétaux se situe entre 0,85 et 31,97 µg de protéine recombinante par gramme de feuilles fraîches. Plusieurs facteurs dont la toxicité et la stabilité des différentes constructions dans les plantes peuvent expliquer cet écart. Dans nos conditions expérimentales, toutes les constructions contenant le fragment fljB ont permis la production d'anticorps spécifiques à fljB dans les souris immunisées avec les extraits végétaux. Par contre, aucune construction n'as permis la production d'anticorps spécifiques à VP7 ou VP4. Ces résultats montrent que l'expression transitoire dans Nicotiana benthaminana permet de produire rapidement de multiples antigènes et que les protéines fortement immunogéniques dans les extraits végétaux induisent une réponse humorale chez les souris. Plusieurs applications de cette technologie sont envisageables dans la cadre du contrôle endémique du RVH ou de Salmonella typhimurium. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Rotavirus humain, VP7, VP4, Salmonella typhimurium, fljB, Expression transitoire, Vaccins à base de plantes, Nicotiana benthaminana.

Nerve agents (NAs) inhibit the essential enzyme acetylcholinesterase. Classified as chemical weapons, NAs are considered a threat to soldiers on the frontlines of warzones. Current treatments can prevent death from NA poisoning, but are not effective in preventing convulsions, seizures, or subsequent brain damage. Butyrylcholinesterase (BChE) binds to NAs, rendering the chemicals harmless to acetylcholinesterase.. Two hundred mg of BChE is the putative prophylactic dose for adult humans, but is difficult to obtain in large quantities from expired human serum. Although recombinant BChE has been expressed in several organisms, the yields are still low. Nicotiana benthamiana is an attractive plant for transient protein production due to its quick growth rate, abundance of tissue, and history of successful recombinant protein production. For this research, N. benthamiana was infiltrated with viral based vectors as well as binary vectors containing the human BChE gene. Multiple assays indicated that binary vector BChE-105-1 + P19 enabled the best expression, producing 26 mg BChE/kg tissue. / Canada Research Chair Program, OMAFRA,

Sonchus yellow net virus (SYNV), Potato yellow dwarf virus (PYDV) and Lettuce Necrotic yellows virus (LNYV) are members of the Rhabdoviridae family that infect plants. SYNV and PYDV are Nucleorhabdoviruses that replicate in the nuclei of infected cells and LNYV is a Cytorhabdovirus that replicates in the cytoplasm. LNYV and SYNV share a similar genome organization with a gene order of Nucleoprotein (N), Phosphoprotein (P), putative movement protein (Mv), Matrix protein (M), Glycoprotein (G) and Polymerase protein (L). PYDV contains an additional predicted gene between N and P, denoted as X, that has an unknown function. In order to gain insight into the associations of viral proteins and the mechanisms by which they may function, we constructed protein localization and interaction maps using novel plant expression vectors. Sub‐cellular localization was determined by expressing the viral proteins fused to green fluorescent protein in leaf epidermal cells of Nicotiana benthamiana. Protein interactions were tested in planta using bimolecular fluorescence complementation (BiFC). All three viruses showed Mv to be localized to the cell periphery and the G protein to be membrane associated. Comparing the interaction maps revealed that only the N‐P and M‐M interactions are common to all three viruses. Associations unique to only one virus include G‐Mv for SYNV, M‐Mv, M‐G, and N‐M for PYDV and P‐M for LNYV. The cognate N‐P proteins of all three viruses exhibit changes in localization when co‐expressed. To complement the mapping data, we also mapped the functional domains in the glycoproteins of SYNV and LNYV. The truncation of the carboxy terminus has no effect on localization compared to the full‐length protein; the nuclear localization signals (NLSs) present in SYNV‐G do not interact with known importins. These data suggest that although the interactions of the three viruses differ, each protein may have similar functional domains.

Techn. University, Diss., 2000--Braunschweig.

The HIV/AIDS pandemic has dramatically altered patterns of morbidity and mortality in sub-Saharan Africa during the last two decades. In the absence of HIV vaccine, microbicides may offer viable option for protection against HIV infection. Microbicides are products that are applied topically inside the vagina or rectum that act to impede transmission of HIV and other sexually transmitted diseases. Small human chemokines such as RANTES (regulated upon activation, normal T cell expressed and secreted) are currently been investigated as microbicides candidates. A number of N-terminally modified RANTES analogues such as 5P12 and 6P4 with a much higher antiviral potency have been developed and they have strong potential for use as microbicides. Since plants offer an alternative option for cost effective production of protein therapeutics, we evaluated the feasibility of expressing 5P12 and 6P4 in Nicotiana benthamiana species. 5P12 is considered the most promising candidate for use in the microbicide pipeline because it inhibits HIV infection through cellular receptor antagonism. Hence its feasibility of expression was also evaluated in Lycopersicon esculentum (tomato). The two analogues were transiently expressed in the selected plant species via agrobacterium-mediated transfection. For expression in N. benthamiana, two different vectors (pTRA and MagnICON) were used to deliver the two analogues for transient expression. About 6-8 weeks-old N. benthamiana plants were agroinfiltrated via needle injection and vacuum infiltration methods and targeted to four subcellular compartments viz: apoplast, chloroplast, cytosol and endoplasmic reticulum (ER). The agroinfiltrated leaves were replanted, grown in a tissue culture laboratory and harvested after different periods. For expression in L. esculentum, the MagnICON constructs were used to deliver the 5P12 gene into four different developmental stages of tomato fruits viz: mature green (MG), breaker (B), pink (P) and ripe (R) via needle injection. The agroinjected tomato fruits were incubated in a dark cupboard and harvested after different periods. xiii Proteins were extracted from the harvested material and evaluated for 5P12 and 6P4 expression. ELISA results showed expression of 5P12 and 6P4 in N. benthamiana leaves which was detectable at 3-9 days post infiltration (dpi). Similar results were obtained for 5P12 and 6P4, consequently only results for 5P12 are reported. The vacuum infiltrated leaves of both pTRA and MagnICON constructs led to higher yields than the needle injected leaves. The highest yields were obtained with the MagnICON constructs. The highest 5P12 expression level of 603 μg/kg fresh weight leaf tissues (~0.024% TSP) was obtained in the apoplast at 9 dpi. The pTRA constructs had the highest expression levels of 0.63μg/kg FW in the cytosol at 3 dpi. 5P12 was also detectable at 3-9 dpi in L. esculentum, based on ELISA results. The highest 5P12 expression of 23.56 μg/kg FW and pH 4.75 tissues was obtained at the MG stage in the apoplast at 9 dpi. Western blot analysis confirmed the size of plantmade 5P12. Moreover, the plant extracts had anti-viral activity and were not toxic to TZM-bl cells. Our results show that the RANTES can be made in both N. benthamiana and L. esculentum and that the levels are not different from other systems reported previously. Furthermore, this is the first report that a chemokine has been expressed in plants. The quantities expressed were low making the commercial development of a microbicide from these species impractical. However, production of bulky leaf material may enhance the quantities. / Thesis (PhD)--University of Pretoria, 2013. / gm2013 / Paraclinical Sciences / unrestricted