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This thesis presents a more in-depth understanding of the interaction between the pathogenic fungus Sporisorium scitamineum and sugarcane, a disease known as \"cane smut\". The development of a long structure like a \"whip\" from the meristem of infected plants is the main characteristic of the disease, allowing the effective dispersion of teliospores in the field. Infected plants have a reduced sucrose content and juice quality, leading to considerable economic losses. In the first chapter, the gene expression profile of the pathogen during its development in planta - in the first moments of infection and after the emission of the whip - and in vitro was evaluated using the RNAseq technique. Were analyzed genes preferentially expressed in each condition, differentially expressed in comparison to its growth in vitro, and expressed only during interaction. The results allowed the identification of some potential pathogenicity mechanisms, active effectors and gene clusters expressed only during interaction. In the second chapter, the transient expression technique was used to determine the target cell compartment of some of the candidate effectors and to establish a viable protocol for the study of S. scitamineum proteins. The four putatively secreted genes most expressed during the initial moments of the interaction were fused to the gene encoding the fluorescent green protein (Citrine) and expressed in Nicotiana benthamiana. The results of confocal microscopy and westernblots indicated an accumulation of each candidate protein in the membrane, cytosol and/or nucleus, in addition to the occurrence of post-translational modifications. These data offer new study opportunities for the identification of plant proteins that interact with such effectors. In the third chapter, the transcriptional responses of sugarcane in the first moments of a compatible interaction and after the development of the whip were analyzed using again the data obtained from the dual RNAseq cane-smut. Among the main responses, was identified an increase in MADS-type transcription factors expression, indicating that the whip development may use a route similar to flowering, whose signaling seems to start as early as the colonization. In addition, whip development is accompanied by increased transcription of genes involved in energetic pathways, and hormones synthesis and signaling pathways. Genes encoding RGAs were differentially expressed and may be related to pathogen effector\'s recognition. In the fourth chapter, the metabolic profile of sugarcane was evaluated during disease progression, confirming that in the meristem of infected plants carbon allocation is channeled to energetic pathways, besides the regulation of several amino acids and changes in plant cell composition in response to whip development. Metabolomics approach also allowed the identification of a probable mycotoxin derived from S. scitamineum. The results obtained in this study contributed to increase the understanding of the interaction between S. scitamineum and sugarcane that is characterized by high complexity and specialization to the host, and can be used in a way to help the characterization of resistant varieties and contribute to the improvement of sugarcane with resistance to smut. / Esta tese apresenta uma compreensão mais aprofundada da interação entre o fungo patogênico Sporisorium scitamineum e a cana-de-açúcar, doença conhecida como \"carvão da cana\". O desenvolvimento de uma longa estrutura similar a um \"chicote\" a partir do meristema de plantas infectadas é a principal característica da doença, permitindo a efetiva dispersão dos teliósporos no campo. As plantas doentes apresentam um teor reduzido de sacarose e qualidade do sumo, levando a perdas econômicas consideráveis. No primeiro capítulo, o perfil de expressão gênica do patógeno durante o seu desenvolvimento in planta - nos primeiros momentos da infecção e após a emissão do chicote - e in vitro foi avaliado utilizando a técnica RNA-Seq. Foram analisados os genes preferencialmente expressos em cada condição, diferencialmente expressos em relação ao crescimento em meio de cultura, ou expressos apenas durante a interação. Os resultados permitiram a elaboração de hipóteses sobre os mecanismos de patogenicidade, sobre os genes candidatos a efetores ativos e a identificação de agrupamentos de genes expressos apenas durante a interação. No segundo capítulo, para determinar o compartimento celular alvo de alguns dos efetores candidatos e estabelecer um protocolo viável para o estudo de proteínas de S. scitamineum foi utilizada a técnica de expressão transiente. Os quatro genes mais expressos durante os momentos iniciais da interação que fazem parte do secretoma do fungo foram fusionados ao gene que codifica a proteína verde fluorescente (Citrina) e expressos em Nicotiana benthamiana. Os resultados de microscopia confocal e westernblots indicaram um acúmulo de cada uma das proteínas candidatas na membrana, citosol e/ou núcleo, além da ocorrência de modificações pós-traducionais. Esses dados oferecem novas oportunidades de estudo para a identificação de proteínas vegetais que interagem com tais efetores. No terceiro capítulo, as respostas transcricionais da cana-de-açúcar nos primeiros momentos de uma interação compatível e após o desenvolvimento do chicote foram analisadas utilizando novamente os dados obtidos a partir do dual RNAseq cana-carvão. Entre as principais respostas da cana destacou-se um aumento da expressão de genes que codificam fatores de transcrição do tipo MADS, indicando que o desenvolvimento do chicote pode usar uma rota semelhante à do florescimento, cuja sinalização parece iniciar logo nos primeiros momentos de colonização. Além disso, o desenvolvimento do chicote é acompanhado pelo aumento da transcrição de genes envolvidos em vias energéticas, e vias de síntese e sinalização hormonal. Genes que codificam para RGAs foram diferencialmente expressos e podem estar relacionados ao reconhecimento de efetores. No quarto capítulo, foi avaliado o perfil metabólico da cana-de-açúcar durante a progressão da doença, confirmando que no meristema de plantas infectadas ocorre um aumento da alocação de carbono em vias energéticas, além da regulação de vários aminoácidos e mudanças em relação à composição da parede celular em resposta ao desenvolvimento do chicote. A abordagem metabólica também permitiu a identificação de uma provável micotoxina derivada de S. scitamineum. Os resultados obtidos neste estudo contribuíram para aumentar a compreensão da interação entre S. scitamineum e a cana-de-açúcar que se caracteriza pela alta complexidade e especialização ao hospedeiro, e poderão ser utilizados de forma a auxiliar a caracterização de variedades resistentes e contribuir para o melhoramento da cana-de-açúcar com resistência ao carvão.

abstract: Gene delivery is a broadly applicable tool that has applications in gene therapy, production of therapeutic proteins, and as a study tool to understand biological pathways. However, for successful gene delivery, the gene and its carrier must bypass or traverse a number of formidable obstacles before successfully entering the cell’s nucleus where the host cell’s machinery can be utilized to express a protein encoded by the gene of interest. The vast majority of work in the gene delivery field focuses on overcoming these barriers by creative synthesis of nanoparticle delivery vehicles or conjugation of targeting moieties to the nucleic acid or delivery vehicle, but little work focuses on modifying the target cell’s behavior to make it more amenable to transfection. In this work, a number of kinase enzymes have been identified by inhibition to be targets for enhancing polymer-mediated transgene expression (chapter 2), including the lead target which appears to affect intracellular trafficking of delivered nucleic acid cargo. The subsequent sections (chapters 3 and 4) of this work focus on targeting epigenetic modifying enzymes to enhance polymer-mediated transgene expression, and a number of candidate enzymes have been identified. Some mechanistic evaluation of these targets have been carried out and discussion of ongoing experiments and future directions to better understand the mechanistic descriptions behind the phenomena are discussed. The overall goal is to enhance non-viral (polymer-mediated) transgene expression by modulating cellular behavior for general gene delivery applications. / Dissertation/Thesis / Doctoral Dissertation Chemical Engineering 2016

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23524号 / 農博第2471号 / 新制||農||1087(附属図書館) / 学位論文||R3||N5355(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 髙野 義孝, 教授 寺内 良平, 教授 吉田 健太郎 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

La sialylation des N-glycanes du fragment Fc des immunogobulines G (IgG) est une modification peu fréquente des IgG humaines. Pourtant, elle est l’objet de beaucoup d’attention depuis que deux articles fondateurs ont été publiés, qui montrent l’un que la sialylation des IgG diminue leur capacité à déclencher la cytotoxicité cellulaire dépendant de l’anticorps (ADCC), et l’autre que les IgG sialylées en α2,6 seraient la fraction efficace des IgG intraveineuses (IgIV) anti-inflammatoires. Les anticorps monoclonaux thérapeutiques, qui sont le plus souvent des IgG recombinantes produites en culture de cellules de mammifère, connaissent depuis la fin des années 90 un succès et une croissance phénoménaux sur le marché pharmaceutique. La maîtrise de la N-glycosylation du Fc des IgG est une clé de l’efficacité des anticorps monoclonaux. Si les IgG sialylées sont des molécules peu fréquentes in vivo, elles sont très rares en culture cellulaire. Dans cette étude, nous avons développé une méthode de production d’IgG avec une sialylation de type humain en cellules CHO. Nous avons travaillé principalement sur la mise au point d’une stratégie de production d’IgG sialylées par co-expression transitoire d’une IgG1 avec la β1,4-galactosyltransférase I (β4GTI) et la β-galactoside-α2,6-sialyltransférase I (ST6GalI). Nous avons montré que cette méthode permettait d’enrichir l’IgG1 en glycane fucosylé di-galactosylé mono-α2,6-sialylé G2FS(6)1, qui est le glycane sialylé présent sur les IgG humaines. Nous avons ensuite adapté cette méthode à la production d’IgG présentant des profils de glycosylation riches en acides sialiques, riches en galactose terminal, et/ou appauvris en fucosylation. L’analyse des profils de glycosylation obtenus par la co-expression de diverses combinaisons enzymatiques avec l’IgG1 native ou une version mutante de l’IgG1 (F243A), a permis de discuter des influences respectives de la sous-galactosylation des IgG1 en CHO et des contraintes structurales du Fc dans la limitation de la sialylation des IgG en CHO. Nous avons ensuite utilisé les IgG1 produites avec différents profils de glycosylation afin d’évaluer l’impact de la sialylation α2,6 sur l’interaction de l’IgG avec le récepteur FcγRIIIa, principal récepteur impliqué dans la réponse ADCC. Nous avons montré que la sialylation α2,6 augmentait la stabilité du complexe formé par l’IgG avec le FcγRIIIa, mais que ce bénéfice n’était pas directement traduit par une augmentation de l’efficacité ADCC de l’anticorps. Enfin, nous avons débuté le développement d’une plateforme d’expression stable d’IgG sialylées compatible avec une production à l’échelle industrielle. Nous avons obtenu une lignée capable de produire des IgG enrichies en G2FS(6)1 à hauteur de 400 mg/L. Cette étude a contribué à une meilleure compréhension de l’impact de la sialylation sur les fonctions effectrices des IgG, et a permis d’augmenter la maîtrise des techniques de modulation du profil de glycosylation des IgG en culture cellulaire. / Only a fraction of the N-glycans present on the Fc fragment of the human IgGs is sialylated. However, a new interest for sialylation has risen since two major articles were published, one showing that sialylation reduces the capacity of the antibody to trigger antibody-dependent cell cytotoxicity (ADCC), whereas the other showed that the IgGs carrying α2,6-sialic acids on their Fc N-glycans were responsible for the anti-inflammatory activity of intravenous immunoglobulins (IVIGs) injected at high doses. Therapeutic monoclonal antibodies (mAbs) are in majority recombinant IgGs produced in mammalian cell culture. Since the end of the nineties, mAbs have become a major class of pharmaceutical products, and their success is still growing. The control of Fc N-glycosylation is a key parameter for the improvement of the therapeutic efficacy of mAbs. Sialylated IgGs are found only as traces in the classic CHO cell culture processes. In this study, we developed a method for the production of IgGs with a human-like sialylation in CHO cells. We focused on a production strategy relying on the transient co-expression of an IgG1 with the β1,4-galactosyltransferase I (β4GTI) and the β-galactoside-α2,6-sialyltransferase I (ST6GalI). We showed that this method allowed the enrichment of the IgG1 glycoprofile in the fucosylated di-galactosylated mono-α2,6-sialylated glycane G2FS(6)1, which is the main sialylated glycan found in human IgGs. We then adapted this method to the production of highly galactosylated or highly sialylated IgGs with and without core-fucosylation. The analysis of the glycosylation profiles obtained using the various enzyme combinations co-expressed with the native IgG1 or the mutant IgG1 F243A allowed us to discuss the influence of the under-galactosylation found in IgGs produced in CHO cells versus the Fc structural constraints on the limitation of IgG sialylation in CHO cells. We used the IgG1 glycovariants produced with our method to assess the impact of Fc α2,6-sialylation on the interaction of the IgG with the receptor FcγRIIIa, which is the main receptor mediating the ADCC response. We showed that the presence of α2,6-sialylation in the Fc increased the stability of the IgG-FcγRIIIa complex. This benefit however did not translate into an improved ADCC capacity. Finally, we initiated the development of a stable expression platform for the production of sialylated IgGs at yields relevant for the industry. We obtained a cell line capable of producing IgGs enriched in G2FS(6)1 at 400 mg/L. This may eventually represent a novel approach to manufacture a recombinant IVIG surrogate. With this work, we contributed to a better understanding of the impact of sialylation on the effector functions of IgGs. We also improved our understanding of the techniques allowing for the modification and control of the glycosylation profile of IgGs in cell culture.

PIN-FORMED (PIN) proteins are plant-specific secondary transporters acting in the efflux of plant signaling molecule auxin from cells. Their asymmetrical localization within cells determines the directionality of auxin flow and thereby influences plant development. The activity of PIN proteins is regulated at multiple levels; however the primary step in the regulation of PIN proteins takes place at the level of gene transcription. Therefore the main focus of this diploma thesis is the characterization of the transcriptional regulation of PIN proteins, namely PIN4 protein. The observation of plants carrying transcriptional fusion consisting of various lengths of PIN4 promoter and green fluorescent protein (GFP) showed which part of PIN4 promoter is essential for binding transcription factors and for the start of transcription. This part of PIN4 promoter was used as bait for transcription factors in yeast one hybrid screens. Altogether, 24 transcription factors were identified in which the most numerous were transcription factors from GATA and APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) families. To verify the interactions between identified transcription factors and PIN4 promoter, the protoplast transient expression assay was used. Protoplasts isolated from Arabidopsis thaliana leaves and tobacco BY-2 cell...

The latest model of tertiary structure of capsid protein of potato virus X (PVX CP) was used as a template to design new insertion sites suitable for the preparation of PVX-based antigen presentation system. Based on this model, seven insertion sites (A-G) located in putative surface loops were tested. As an antigen inserted into these sites was used 17 amino acids long epitope derived from human papillomavirus type 16 E7 oncoprotein (E7 epitope) fused with either 6xHis tag or StrepII tag in both possible orientations (6xHis-E7 and E7-6xHis, StrepII-E7 and E7-StrepII). Prior to plant expression, modified PVX CPs were expressed in Escherichia coli MC1061. The results showed that only PVX CP carrying StrepII-E7 or E7-StrepII in the insertion site A formed virus particles. The results from transient expression experiments with modified PVX CPs in Nicotiana benthamiana showed that only the insertion site A (located between 24th and 25th amino acid in the PVX CP) could tolerate all tested inserts. Importantly, viral particles were detected only in the presence of StrepII tag and their stability was affected by the insert orientation (StrepII-E7 vs. E7-StrepII) as only the viral particles presenting E7-StrepII could be purified. Besides the preparation of PVX-based antigen presentation system, an...

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Thesis (MSc (Genetics))--University of Stellenbosch, 2011. / Includes bibliography / ENGLISH ABSTRACT: This study investigated the use of antimicrobial peptides (AMPs) as possible source of resistance against a range of pathogens in grapevine. Whilst the ultimate aim would be to express AMPs in grapevine, the development of transgenic grapevine is time consuming and therefore pre-screening of potential AMPs is necessary. These small molecules, of less than 50 amino acids in length, are expressed by almost all organisms as part of their non-specific defence system. In vitro pre-screening of AMP activity is valuable but is limited since the activity on artificial media may differ from the AMP activity in planta. These tests are also restricted to pathogens which can be cultured in vitro. These limitations can be overcome by using transient expression systems to determine the in planta activity of AMPs against pathogens of interest. In this study transient systems were used to express AMPs in developed plant tissue to test their efficacy against grapevine pathogens such as Agrobacterium vitis, Xylophilus ampelinus and aster yellows phytoplasma. Aster yellows phytoplasma, which was recently discovered in local vineyards, is known to cause extensive damage and therefore pose a great threat to the South African grapevine industry. To study the in planta effect of AMPs against the abovementioned pathogens, transient expression vectors were constructed expressing either of the AMPs D4E1 or Vv-AMP1. D4E1 is a synthetically designed AMP known to be active against bacteria and fungi, while Vv-AMP1, isolated from grapevine berries, has already shown activity against fungi. In a transient approach in grapevine, the expression of foreign genes from viral and non-viral vectors was confirmed by expression of the marker genes β-glucuronidase and Green Fluorescent Protein, while tissue-printing immunoassays confirmed viral replication and systemic spread in Nicotiana benthamiana. The viral vectors were based on the phloem-limited virus grapevine virus A. Only Agrobacterium-mediated 35S transient expression vectors were used for AMP in planta activity screening since the viral-mediated expression in grapevine was insufficient for screening against A. vitis and X. ampelinus as it was restricted to phloem tissues after whole-leaf infiltration. No phytoplasma-infected material could be established and as a result AMP activity screening was only performed against the A. vitis and X. ampelinus. Quantification of the bacteria was performed by qPCR. Vv-AMP1 did not show activity against either of the two bacteria in planta while D4E1 was found to be active against both. The observed in planta activity of D4E1 correlated with the in vitro activity as measured in an AMP plate bioassay. In contrast to in vitro screenings, the in planta AMP activity screening might give a more accurate representation of the potential antimicrobial activity of the peptide in a transgenic plant environment. This study proved that transient expression systems can be used as a pre-screening method of AMP activity in planta against grapevine pathogens, allowing the screening of various AMPs in a relatively short period of time before committing to transgenic grapevine development. / AFRIKAANSE OPSOMMING: Hierdie studie het die gebruik van antimikrobiese peptiede (AMPe) as 'n moontlik bron van weerstand teen 'n reeks van patogene in wingerd ondersoek. Alhoewel die uiteindelike doel sal wees om AMPe uit te druk in wingerd, is transgeniese wingerd ontwikkeling tydrowend en daarom is vooraf evaluering van potensiële AMPe nodig. Hierdie klein molekules, van minder as 50 aminosure in lengte, word uitgedruk deur amper alle organismes as deel van hul nie-spesifieke verdedigingsisteem. In vitro vooraf evaluering van AMP aktiwiteit is van waarde, maar is beperk aangesien die aktiwiteit op kunsmatige media mag verskil van die AMP-aktiwiteit in planta. Hierdie toetse is ook beperk tot patogene wat in vitro gekweek kan word. Hierdie beperkinge kan oorkom word deur gebruik te maak van tydelike uitdrukkingsisteme om die in planta aktiwiteit van AMPe te bepaal teen patogene van belang. In hierdie studie is tydelike uitdrukkingsisteme gebruik om AMPe uit te druk in ontwikkelde plantweefsel om hul effektiwiteite te toets teen wingerdpatogene soos Agrobacterium vitis, Xylophilus ampelinus en aster yellows fitoplasma. Aster yellows fitoplasmas, wat onlangs in plaaslike wingerde ontdek is, is bekend vir die uitgebreide skade wat hul aanrig en hou daarom 'n groot bedreiging in vir die Suid-Afrikaanse wingerd industrie. Om die in planta effek van AMPe teen die bogenoemde patogene te bestudeer is tydelike uitdrukkingsvektore ontwikkel wat die AMPe D4E1 of Vv-AMP1 uitdruk. D4E1 is 'n sinteties-ontwerpte AMP wat aktief is teen bakterieë en fungi, terwyl Vv-AMP1, wat uit druiwekorrels geïsoleer is, alreeds aktiwiteit teen fungi getoon het. In 'n tydelike uitdrukkingsbenadering in wingerd is die uitdrukking van transgene, vanaf virus of nie-virus gebaseerde vektore, bevestig deur die uitdrukking van die merker gene β-glukuronidase en die Groen Fluoresserende Proteïen, terwyl weefsel afdrukkings-immunotoetse virus replisering en sistemiese beweging in Nicotiana benthamiana bevestig het. Die virusvektore was gebaseer op die floëem-beperkte virus, wingerdvirus A. Slegs Agrobacterium-bemiddelde 35S tydelike uitdrukkingsvektore is gebruik om die AMP in planta aktiwiteit te bepaal aangesien die virus-bemiddelde uitdrukking in wingerd onvoldoende was vir evaluering teen A. vitis en X. ampelinus weens die beperking tot die floëem weefsel na infiltrering van die totale blaar. Geen fitoplasma geïnfekteerde materiaal kon gevestig word nie, en daarom is AMP aktiwiteitsevaluering slegs teen A. vitis en X. ampelinus uitgevoer. Kwantifisering van die bakterieë is deur middel van qPCR uitgevoer. Vv-AMP1 het geen aktiwiteit getoon teen enige van die bakterieë in planta nie, terwyl D4E1 aktief was teen beide. Die waargenome in planta aktiwiteit van D4E1 het ooreengestem met die in vitro aktiwiteit soos bepaal deur 'n AMP plaat bio-toets. In kontras tot in vitro evaluering kan die in planta AMP-aktiwiteit evaluering 'n meer akkurate voorspelling bied van die potensiële antimikrobiese aktiwiteite van die peptied in 'n transgeniese plant omgewing. Hierdie studie het bewys dat tydelike uitdrukkingsisteme gebruik kan word as 'n voorafgaande evalueringsmetode vir AMP in planta aktiwiteit teen wingerdpatogene, wat die evaluering van 'n verskeidenheid AMPe in 'n relatiewe kort tydperk toelaat voor verbintenis tot die ontwikkeling van transgeniese wingerd.

Potato is one of the main targets for genetic improvement by gene transfer. The aim of this study was to compare the efficacy of genetic transformation of potato, cultivar Bintje, using two methods: Agrobacterium tumefaciens mediated transformation and microprojectile bombardment. The same plasmid p35SGUSint, which cosists of 35S CaMV promoter, gus and nptII genes, was used for both transformations of internodal potato explants. Kamamycin selection, transient and stable expressions of {$\beta$}-glucuronidase and PCR amplification of gus and nptII transgenes were used for transgenic plant selection, identification and analysis.